Developmental regulation of N-terminal H2B
methylation in Drosophila melanogaster
Ana Villar-Garea1, Ignasi Forne1, Irene Vetter1, Elisabeth Kremmer2,
Andreas Thomae1and Axel Imhof1,*
1Munich Center of Integrated Protein Science and Adolf-Butenandt Institute, Ludwig Maximilians University of
Munich, 80336 Munich and2Helmholtz Zentrum Munich, Institute of Molecular Immunology, 81377 Munich,
Received July 19, 2011; Revised October 8, 2011; Accepted October 12, 2011
Histone post-translational modifications play an im-
portant role in regulating chromatin structure and
gene expression in vivo. Extensive studies investi-
gated the post-translational modifications of the
core histones H3 and H4 or the linker histone H1.
Much less is known on the regulation of H2A and
H2B modifications. Here, we show that a major
modification of H2B in Drosophila melanogaster is
the methylation of the N-terminal proline, which in-
creases during fly development. Experiments per-
formed in cultured cells revealed higher levels of
H2B methylation when cells are dense, regardless
of their cell cycle distribution. We identified dNTMT
(CG1675) as the enzyme responsible for H2B methy-
lation. We also found that the level of N-terminal
methylation is regulated by dART8, an arginine
methyltransferase that physically interacts with
dNTMT and asymmetrically methylates H3R2. Our
results demonstrate the existence of a complex con-
taining two methyltransferases enzymes, which
negatively influence each other’s activity.
In the eukaryotic nucleus DNA is packaged into chroma-
tin by its association with the basic core histones. The
folding of chromatin fibers has a major impact on many
aspects of nuclear function and is regulated by the post-
translational modification of the histone tail domains. In
multicellular organisms malfunction of the enzymatic
machinery that establishes these modifications leads to
abnormalities such as failures in embryonic development,
cancer and other diseases (1–4). Histone modifications
have been shown to mark specific chromosomal domains
and serve as an indexing system of the genome to distin-
guish transcriptionally active from inactive regions (5–9).
In addition, they also play a role during histone deposition
(10–14) where an ordered appearance and removal of
distinct modifications is required for proper chromatin
assembly. The modifications on the histone N-terminal
tails are recognized by specialized proteins that selectively
bind modified histones (15,16). The specific binding to
particular modifications can then either lead to structural
changes of chromatin or recruit enzymatic activities to
specific loci, which in turn can either stimulate or inhibit
a subsequent modification. Examples of this phenomenon
are the stimulation of the acetylation of H3K14 by a phos-
phorylation of H3S10 (17,18), the inhibition of H3K4
methylation by an adjacent dimethylation of H3R2
(19,20) or the inhibition of H3K4 demethylation by the
phosphorylation of H3T6 (21). The two modifications that
influence each other do not have to reside on the same
moleculeas it has been
ubiquitination of H2B by Rad6 facilitates the methylation
of H3K4, suggesting a crosstalk of the two histone tails
(22,23). Another example for such a crosstalk is the phos-
phorylation of H3S10 by the Pim1 kinase, which stimu-
lates the acetylation of H4K16 (24).
Most of the global histone modification analyses done
so far were performed on the two core histones H3 and H4
whereas the post-translational modifications of canonical
H2A and H2B have been less well studied in metazoa.
Only the ubiquitination of H2A and H2B has been sug-
gested to have a specific function such as the silencing of
genes (25) or the stimulation of H3K4 and H3K79 methy-
lation, respectively (22,23,26). Human H2B is phosphory-
lated at S14 by the caspase cleaved mammalian Mst-1
kinase (27). This phosphorylation has been proposed to
mediate chromatin condensation during apoptosis (27),
which is counteracted by the acetylation of the adjacent
K15 (28). H2A is phosphorylated at S1 (29) but so far this
*To whom correspondence should be addressed. Tel: +49 89 218075420; Fax: +49 89 218075440; Email: Imhof@lmu.de
Ana Villar-Garea, Institute for Biochemistry, Genetic and Microbiology, University of Regensburg, 93053 Regensburg, Germany.
Nucleic Acids Research, 2012, Vol. 40, No. 4Published online 3 November 2011
? The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
has not been shown to be regulated (29,30). The analysis
of H2A and H2B methylation and acetylation in higher
eukaryotes by mass spectrometry (MS) has been severely
hampered by the multitude of different isoforms with simi-
lar molecular masses making it difficult to distinguish
post-translational modifications from sequence variants
(31). Drosophila melanogaster, in contrast to most higher
organisms, has just a single H2B variant, which greatly
facilitates the analysis of H2B PTMs.
Drosophila melanogaster H2B is ubiquitinated at K120
(32,33), phosphorylated at S33 (34) and methylated at the
N-terminal proline (35). Although the methylation of the
terminal a-amino group has been found in H2B of a
variety of organisms and a number of proteins other
than histones (36), its biological significance is largely un-
known. Drosophila tissue culture cells show an increased
proline methylation in response to heat shock and arsenite
treatment (35,37). More recently, the N-terminal methyla-
tion of mammalian RCC1 has been shown to be crucial
for its binding to chromatin and for proper mitotic
chromosomal segregation (38). A pair of ortholog enzymes
that perform N-terminal methylation of proteins in
humans and yeast, NRMT (METTL11A) and YBR261C/
Tae1, respectively, has been also isolated (36,39).
Here, we show that N-terminal methylation of H2B in
D. melanogaster is not only regulated by cellular stress
such as heat shock but also changes during development.
In tissue culture cells the proportion of methylated histone
depends on cell density, but not on the cell cycle distribu-
tion. Knockdown experiments and in vitro assays demon-
strate that Drosophila’s ortholog of NRMT, dNTMT
(CG1675) mono- and di-methylates the N-terminus of
H2B. Interestingly, dNTMT forms a complex with an
arginine-specific H3 methyltransferase dART8, which
targets H3R2. Although the interaction has no effects on
the activity of the enzymes in vitro, modulation of dART8
levels in cells has a substantial effect on H2B N-terminal
methylation in vivo. A knockdown of dART8 results
in an increase of H2B methylation whereas over-
expression leads to a reduction, suggesting a repressive
effect of dART8 levels on the modification of the H2B
MATERIALS AND METHODS
SL2 and Kc cells were maintained at 26?C in Schneider’s
Drosophila medium with glutamine (GIBCO) supple-
mented with 10% heat-inactivated FBS (Sigma) and
50U/ml penicillin and 50mg/ml streptomycin (CC Pro,
Oberdorla, Germany). Except otherwise stated, cells
were kept exponentially growing by diluting the cultures
every 2–3 days with fresh medium to a density of 0.5?106
cells/ml. For each experiment, cells were harvested by cen-
trifugation of the suspensions at 500g during 5min (r.t.)
and decanted. Then, the same number of cells per sample
(typically, 4–8?106cells) was seeded with a volume-to-
surface ratio of 0.24ml/cm2.
Cell density and heat shock experiments
Exponentially growing cells were collected as indicated
above, resuspended in fresh medium at the indicated
densities and cultured for further 24h (except otherwise
stated) before harvesting. For heat shock experiments, ex-
ponentially growing cells were incubated at 37?C for 1h.
Histones were isolated immediately after heat shock.
One to two million cells per sample were harvested, washed
once with PBS, resuspended in 0.17ml ice-cold PBS and
fixed by addition of 0.33ml ice-cold absolute ethanol
for 30min. After fixation, each sample was washed
twice with PBS, resuspended in 0.5ml PBS and treated
with 50mg RNAse A for 15min. After cooling on ice,
DNA was stained with 2mg propidium iodide (Sigma).
cytometer (BD Biosciences) equipped with a 488nm
laser. Typically, 3?104events per sample were acquired.
ina FACS Canto
Sodium butyrate (Merck) was dissolved in milliQ water
(2.0mM final concentration) and rapamycin (Sigma) was
reconstituted in ethanol (10mM final concentration). Both
solutions were sterile filtered (0.22mm), aliquoted and
stored at ?20?C. For the experiments, cells were harvested,
seeded in fresh medium (Kc cells: 0.5?106cells/ml; SL2
cells 1?106cells/ml) and treated with the indicated con-
centration of the chemical or the solvent only. Histones
were isolated at the indicated time points after drug
Histone extraction from cultured cells
Histone acid extraction was performed as previously de-
scribed (40) withminor
4–8?106cells were harvested as indicated above and sub-
pH=7.5, 10mM NaCl, 3mM MgCl2). If necessary, at
this point samples were stored at ?20?C. Washed pellets
were resuspended in 0.5ml, 0.5M HCl and rotated at 4?C
for 1h. Solutions were cleared by centrifugation (13000g?
5min?4?C) and dialysed against 0.1 M acetic acid at 4?C
(MWCO 6000–8000). Dialysates were freeze-dried and
reconstituted in 50mM Tris pH=6.8 containing 5mM
tris(2-carboxyethyl)phosphine hydrochloride (Pierce) by
using 6.2ml buffer per million cells. For the isolation of
histones using MNase, nuclei were isolated from SL2 cells
using a nuclear extraction buffer (1?PBS, 0.3% Triton
X?100) and digested using 500mU of MNase (Roche)
per 107cells for 10min at 26?C. To isolate chromatin,
the MNase reaction was quenched using 0.5 vol of 0.5M
EGTA and nuclear debris was centrifuged for 20min at
800g. The released chromatin fibers were harvested and
stored at 4?C.
Histone acid extraction from animals
All the samples were snap-frozen in liquid nitrogen imme-
diately after collection. Embryos 0–24h after egg laying
were washed, dechorionated according to (40). For later
Nucleic Acids Research,2012, Vol.40, No. 41537
stages 50–100 unsorted larvae and 30–40 adult flies were
pooled. Histones from frozen animals and frozen de-
chorionated embryos were grinding the frozen samples
in either hydrochloric acid (0.5 M HCl, 5mM DTT) or
by sulfuric acid (0.2 M H2SO4, 1mM DTT). After extrac-
tion histones were either dialysed against 0.1M acetic acid
(HCL extraction) or precipitated by addition of 10 vol
acetone and incubation at 4?C overnight (sulfuric acid
extraction). Lyophilized samples were reconstituted in
300mL 1?Laemmli buffer whereas the acetone precipi-
tated histones were reconstituted in 50ml (sulfuric acid
extraction) 50mMTris pH=6.8
Histones from staged embryos
Nuclei from staged embryos (2–3h, 3–6h, 6–9h and 9–12
after egg laying) were prepared as described before (41)
and histones were extracted with 3 vol nuclear pellet of 0.4
MH2SO4. Extracts were dialysed as indicated above,
freeze-dried and reconstituted in 20mM Tris pH=6.8
containing 1mM DTT.
H2B isolation and MS
Proteins in the extracts were separated by SDS–PAGE
18% acrylamide. After electrophoresis, gels were stained
with Coomassie Brilliant Blue G250 (Merck) and bands
corresponding to H2B were excised. Gel bands were de-
stained, acylated and digested with trypsin as previously
described (42). Alternatively, once the bands were de-
(Roche) at 25?C over night using 0.1 M Tris pH=8.5
as reaction buffer. The reaction products were desalted
with ZipTipm-C18 (Millipore) mixed with a saturated
a-cyano-hydroxycinnamic acid (Sigma) solution and im-
mediately spotted onto a stainless steel target plate.
Spectra were acquired on a Voyager DE STR workstation
(Applied Biosystems). Integration of the signals corres-
ponding to each peptide (isotopic cluster area) was auto-
matically performed by the Data Explorer software
(Applied Biosystems) excluding all the peaks whose
signal-to-noise ratio was smaller than 4 (8,9).
For MS/MS analysis a third of the digest was typically
injected in an Ultimate 3000 HPLC system (LC Packings
Dionex). Samples were desalted on-line by a C18 micro
column (300mm i.d.?5mm, packed with C18 PepMapTM,
5mm, 100A˚by LC Packings), and peptides were separated
with a gradient from 5% to 60% acetonitrile in 0.1%
formic acid over 40min at 300 nl/min on a C18 analytical
column (75mm i.d.?15cm, packed with C18 PepMapTM,
3mm, 100A˚by LC Packings). The effluent from the HPLC
was directly electrosprayed into the LTQ Orbitrap mass
spectrometer (Thermo Fisher Scientific). The MS instru-
ment was operated in the data-dependent mode to auto-
matically switch between full scan MS and MS/MS
acquisition. Survey full scan MS spectra (m/z 300–800
for H2B N-termini in Figure 1; m/z 572–590 H2B
N-termini in Supplementary Figure S2; m/z 300–2000 for
H3 in Figure 6) were acquired in the Orbitrap with
resolution R=30000 (Figure 1 and Supplementary
Figure S2) or 60000 (Figure 6) at m/z400. The three
most intense peptide ions with charge states between two
and five were sequentially isolated (window=2m/z) to a
target value of 10000 and fragmented in the linear ion
trap by collision-induced dissociation (CID). Fragment
ion spectra were recorded in the Orbitrap part of the in-
strument. For all measurements with the Orbitrap
(m/z=371.10123, 445.12002, 519.13882) were used for in-
ternal calibration as described (43). Typical mass spectro-
metric conditions were: spray voltage, 1.4kV; no sheath
and auxiliary gas flow; heated capillary temperature,
200?C; normalized collision energy, 35% for CID in
linear ion trap. The ion selection threshold was 10000
counts for MS2. An activation q=0.25 and activation
time of 30ms were used.
ions from ambientair
Protein cloning and expression of Flag-dNTMT
Flag-dNTMT (accession number NP_610528) was amp-
lified from the FMO4789 expression plasmid using oligo-
nucleotides: fw: 50-TTTGAATTCGATTACAAGGATGA
GCTTTCAGAC (FLAG tag in bold); rev: TTTAAGCTT
CTATTCCTTGGAGACGGGTTTGCAGG. The PCR
product was cloned into pET28a (Novagen) using unique
EcoRI and HindIII sites. For expression in SL2 cells we
have used the pMCFHBD (FMO4789) vector that was
acquired from the DGRC gold collection. Full length
Drosophila dART8 (accession number NP_609478) was
amplified and cloned into pETG-N-GST (gift of A.
Baiker, University of Munich) for bacterial expression
and in pMT-Flag-HA [derived from pMT-HA (44)] for
expression in SL2 cells. Primer sequences for expression
constructs and knockdowns of dARTs and dNTMT are
available upon request.
Drosophila L2-4 cells (Schneider S2 derivative) were tran-
siently transfected with the FMO4789 dNTMT expression
plasmid (DGRC) using Effectene (Qiagen) according to
the manufacturer’s instructions. Expression was induced
2 days after transfection by the addition of copper sulfate
(0.25mM final). After an additional 48h cells were fixed
with formaldehyde, permeabilized with Triton X?100,
incubated with anti-FLAG M2 antibody (Sigma). After
incubation with fluorescently labeled anti-mouse second-
ary antibody and DNA counterstaining with ToPro-3,
cells on coverslips were mounted with Slow Fade Gold
reagent (Invitrogen). Images were recorded using a Zeiss
510 LSM confocal microscope and processed using
Coimmunoprecipitation of dART8 and dNTMT
SL2 cells stably expressing N-terminal FLAG-HA-tagged
dART8 under a metallothioneine promoter were induced
with 0.25mM copper sulfate 24h before harvest. Cells
were lysed in hypotonic buffer by the addition of Triton
X-100 at a final concentration of 0.02%. After lysis, KCl
concentration was raised to 110mM and proteins were
1538 Nucleic Acids Research, 2012,Vol.40, No. 4
extracted by the addition of ammonium sulfate (1/10th
volume of a 4 M stock). After centrifugation the super-
natant was dialysed against a 150mM KCl buffer, precipi-
tate was removed by centrifugation yielding the extract
used for coimmunoprecipitation. Extract from the equiva-
lent of 5?108cells was used for each immunopre-
cipitation. A mix of rat monoclonal antibodies of the
subtype IgG2a specific for dNTMT (clones 3F4, 4B9,
9E5) was used for dNTMT IPs. A rat monoclonal anti-
body of equal subtype specific for the Xenopus laevis
Smad Interacting Protein was used as a negative control.
Sigma mouse anti-FLAG M2 agarose was used to precipi-
tate FLAG-HA-dART8. The immunoblot was also per-
formed with the above mentioned antibodies.
Figure 1. H2B in D. melanogaster is methylated at the N-terminus. (A) Sequence of the fruit fly’s protein H2B. Open triangles show the cutting sites
for the protease Asp-N, whereas the filled triangles label the cutting sites for trypsin after the free amino groups of the sample have been blocked by
treatment with an anhydride (either D6-acetic or propionic anhydride). (B) MALDI-TOF spectra of digested H2B isolated from SL2 cells. The left
panel shows the signals corresponding to the peptide 1–22, which are obtained after Asp-N digestion of H2B. The right panel displays the signals
corresponding to the peptide 1–28, obtained after treatment of the protein with D6-acetic anhydride followed by trypsin digestion. un, unmodified
proline; Pme1, monomethylated proline; Pme2, dimethylated proline; Ac1, one acetylated lysine. (C) Tandem MS spectra of the peptide 1–22
demonstrate that the observed mono- (upper panel) and dimethylation (lower panel) take place at P1. The mass of the parent ions and the
fragment ions were measured with a resolution of 30000 and 15000, respectively. The inserts display the signals of the corresponding parent
ions and the errors respect to the expected (calculated) values.
Nucleic Acids Research,2012, Vol.40, No. 4 1539
H2B is mono and dimethylated at the N-terminal proline
To determine the modifications present in H2B from D.
melanogaster, the protein was isolated from asynchron-
ously growing SL2 and Kc cells, separated using SDS–
PAGE, digested and analyzed by MS. As H2B is rich in
arginine and lysine residues, we either used trypsin after
acylation of lysine residues (42) or Asp-N as proteases
of choice (Figure 1A). By the combination of the two pro-
teases we could achieve a sequence-coverage of 95%
(Table 1). We clearly detected a large proportion of the
N-terminal peptide generated by an Asp-N or trypsin after
acylation (MH+=2253.34 for Asp-N and 3532.22 for
trypsin after acetylation with D6-acetic anhydride) in a
mono- or dimethylated form (Figure 1B).
Additionally, a part of the N-terminal tail is mono or
diacetylated (Figure 1B). In order to determine the exact
positions of the modifications, Asp-N digested H2B was
submitted to a MS/MS analysis. Fragmentation patterns
allow the assignment of the mono and dimethylation to
position 1, which is in agreement with previous reports
that described a methylation of the N-terminal proline
Drosophila (35) (Figure 1C). The acetylation resides pre-
dominantly at position K11 (mono) or K11 and K17 (di)
with minor acetylations at K10 and 14 (Supplementary
Methylation of H2B increases during development
In order to study the biological function of H2B
N-terminal methylation we measured its level during dif-
ferent developmental stages of D. melanogaster. We
isolated and analyzed the modification pattern of H2B
from embryos at different time points after egg laying
(a.e.l.), 3rd instar larvae and adult flies (Figure 2).
Interestingly, we observed mostly unmodified H2B in
embryos (0–24h a.e.l.) whereas in adult animals mono-
as well as dimethylations were very abundant (Figure 2A
and B). This finding suggests a relationship between H2B
methylation and cell proliferation, senescence or differen-
tiation. We therefore wondered whether levels of H2B
N-terminal methylation are indicative of the proliferative
capacity or the differentiated state of cells.
The methylation pattern in cultured cells depends on cell
To investigate this, we measured the N-terminal methyla-
tion of H2B in Drosophila tissue culture cells that were
initially established from late embryos (45,46). Due to
their embryonic origin we expected H2B from these cell
lines to have a low level of methylation. However, when
we analyzed H2B from SL2 or Kc cell lines we consistently
detected a higher proportion of methylated H2B mol-
ecules compared to the developmental stage they were
derived from (compare Figure 2 and Figure 3). This
suggests that higher amounts of H2B methylation either
reflect an adaptation to tissue culture conditions or an out-
growth of a highly methylated minor cell clone during the
establishment of the cell line. In culture, the proportion of
methylated H2B depends on the cell density, as the methy-
lation levels increase when cells are seeded at higher
densities (Figure 3A). As SL2 and Kc cells accumulated
in G2/M when seeded at such high densities (Figure 3B
and data not shown) we wondered whether we could
simulate the increase in methylation by treatment of the
cells with rapamycin, a drug that inhibits the highly
conserved nutrient-sensing TOR signaling pathway (47)
and led to a similar effect on the cell cycle distribution
of Kc and SL2 cells (Figure 3D and data not shown).
Although the cell cycle distribution was clearly affected
under the conditions we used (Figure 3D), we could not
observe significant changes in the methylation pattern of
H2B (Figure 3C) even after an extended period of treat-
ment. These experiments suggest that neither the cell cycle
arrest nor inhibition of TOR signaling is sufficient to alter
the N-terminal methylation of H2B. It had been shown
previously that the methylation of the N-terminal proline
is stimulated by heat shock (35). To check if this is also the
case in our system, we subjected SL2 cells to a 1-hr heat
shock at 37?C, isolated the histones via acid extraction
and analyzed the levels of H2B N-terminal methylation.
In accordance with data previously published (35,37) we
detected an increase of H2B methylation after heat shock,
which is similar to the one seen when growing cells at
higher density. In summary, we concluded from these ex-
periments that the N-terminal methylation of H2B is able
to integrate several external signals such as proliferative or
dNTMT (CG1675) is the enzyme responsible for
We next asked which enzyme is responsible for the
N-terminal methylation of H2B in Drosophila. Recently
the enzyme that methylates the N-terminus of RCC1
was identified in human and yeast (36,39) and therefore
the Drosophila ortholog (CG1675) was a good candidate
Table 1. Predicted and detected peptide fragments of dH2B after digestion with proteases
me1, me2, ac1, ac2 1–28
1540Nucleic Acids Research, 2012,Vol.40, No. 4
for carrying out this function in the fly. To test the hy-
pothesis, we expressed the protein in bacteria and tested its
ability to methylate recombinant H2B in vitro (Figure 4).
When incubated with radioactive S-adenosyl-methionine
and different recombinant histone molecules, the enzyme
specifically modified H2B but not the other three histones
(Figure 4A). In order to better characterize the enzyme
we determined the appearance of mono and dimethylated
dH2B during the dNTMT catalyzed reaction (Figure 4B).
The transient accumulation of monomethylated H2B
quickly after the reaction had been started suggests that
the enzyme is not processive. However, a more detailed
analysis would be necessary to exclude other explanations
for this observation. As the N-terminal methionine is only
partially cleaved when the protein is produced in bacteria,
a portion of the H2B molecules we have used in the assay
still carried it at the N-terminus. The H2B that still con-
tained the methionine was not methylated by CG1675
(Figure 4C) whereas the one carrying a proline residue
was, suggesting that the enzyme uses the N-terminal
proline as its substrate. This is in good agreement with
the recognition site for the mammalian orthologs, which
has been reported to be (Ala/Ser/Pro)-Pro-Lys (39).
Within Drosophila, 36 proteins carry such a recognition
site (Table 2), which is a similar amount compared to what
has been reported for mammals (39). Surprisingly only
eight of them are evolutionary conserved. Most notably,
all ribosomal subunits that have been suggested to serve as
substrates for NRMT1 were also potential targets in the
fruit fly. Drosophila RCC1, however, has a different
N-terminal sequence making it unlikely as a substrate for
CG1675. Therefore we termed CG1675 dNTMT for
Drosophila N-terminal methyltransferase. Aknockdownof
dNTMT in tissue culture cells leads to a substantial de-
crease of H2B methylation, suggesting that it is a major en-
zyme responsible for the modification in cells (Figure 4D).
When expressed in SL2 cells, tagged-dNTMT mainly lo-
calizes to the nucleus (Figure 5A) pointing to a function in
this organelle. Unfortunately the low transfection effi-
ciency of <10% did not allow us to detect an increased
level of total H2B isolated from a pool of transfected cells.
However, the model of dNTMT acting mainly in the
nucleus is further supported by our finding that over
95% of chromatin-bound H2B is methylated (Figure 5B
and C) whereas we consistently detect about 15–18% of
acid extracted (total) histones in an unmethylated form
dART8 interacts with dNTMT and regulates H2B
In order to get more insights into the function of the
methylation of H2B, we studied the possible regulation
of dNTMT. Yeast two-hybrid screenings (48) had reported
seven interaction partners for this methyltransferase:
CG1324 and Zasp66, whose molecular function is
unknown; Cpr47Ef, a structural protein of the cuticle;
3445.03490.43535.8 3581.2 3626.63672.0
Figure 2. The methylation pattern changes during development. MALDI-TOF spectra of the peptide 1–28 of H2B isolated from a variety of
developmental stages. Prior to MS, histone H2B was treated with deuterated acetic anhydride and subsequently digested with trypsin. (A)
Methylation levels of H2B in 0–24h after egg laying (a.e.l.) embryos (upper panel), 3rd instar larvae (middle panel) and unsorted adult flies
(bottom panel). (B) Methylation levels in staged embryos do not significantly change in the first hours a.e.l. From top to bottom, 2–3, 3–6, 6–9
and 9–12h a.e.l. un, unmodified proline; Pme1, monomethylated proline; Pme2, dimethylated proline.
Nucleic Acids Research,2012, Vol.40, No. 4 1541
SL2 + heat shock
DNA content (PI)
DNA content (PI)
Figure 3. Cell density and heat shock, but not rapamycin influence the methylation levels. MALDI-TOF spectra showing the region of the peptide
1–28 of H2B after treatment with deuterated acetic anhydride and trypsin digestion (A, C and E) and cell cycle histograms (B) and (D) of the
corresponding samples. (A) and (B) Kc cells were seeded in fresh medium at 0.5 (upper) and 4.0?106(lower) cells/ml, collected 24h later and used
either for H2B or cell cycle analysis. (C) and (D) Kc cells were seeded in fresh medium at 0.5?106cells/ml, 24h later were treated with 20nM
rapamycin, harvested at the indicated time points (0, 2 and 24h) and prepared for subsequent analyses. (E) SL2 cells were incubated for 1h at 37?C
and subsequently harvested. The upper panel shows the result for the mock-treated sample and the lower, for the heat shocked one. un, unmodified
proline; Pme1, monomethylated proline; Pme2, dimethylated proline.
1542Nucleic Acids Research, 2012,Vol.40, No. 4
sec24, a cytoplasmatic protein; CG36329, a putative ATP-
dependent peptidase; CG6745, which has putative pseudo-
uridine synthase activity; and dART8, a putative arginine
methyltransferase. We therefore decided to investigate a
possible effect of dART8 levels on H2Bmethylation.
When we knocked down dART8 using specific dsRNA,
we reproducibly observed an increase in H2B dimethyla-
tion. This effect was highly specific for dART8, as a knock
down of other members of this family of arginine
methyltransferases did not influence H2B methylation
levels (Figure 6A). Drosophila Art8 had not been shown
to have arginine methyltransferase activity. We therefore
tested whether the Drosophila protein has a similar speci-
ficity to its closest human ortholog PRMT6, which methy-
lates R2 in histone H3. We expressed dART8 in bacteria
as a GST-fusion protein and performed an in vitro methyl-
transferase assay using the four recombinant core histones
(Supplementary Figure S2A). As expected dART8 mainly
0 25 50 75 100
% of total H2B
Reaction Time (min)
3558.03607.8 3657.63707.4 3757.23807.0
35003552 3604 3656 37083760
Figure 4. dNTMT methylates P1 of H2B in vitro and in vivo. (A) After incubation of different recombinant core histones (H3, H2B, H2A and H4)
with recombinant dNTMT in the presence of3H-S-Adenosyl Methionine (SAM), only H2B became methylated. Upper panel, Coomassie stained
protein gel; lower panel, corresponding autoradiography. (B) In vitro methylation kinetics of dH2B N-terminus by dNTMT. Open circle, unmodified
proline; open triangle, monomethylated proline; filled square, dimethylated proline. N=3. (C) MALDI-TOF spectra of Asp-N digested H2B shows
that the N-terminal peptide becomes methylated after incubation of the histone with dNTMT and SAM (right panel) but not after incubation with
the enzyme alone (left). (D) MALDI-TOF spectra of propionylated, trypsin digested H2B reflect the loss of methylation upon knockdown of
dNTMT. Upper panel: control (GST RNAi); middle and lower panel: knockdown of dNTMT, using two different dsRNA molecules. un, unmodified
proline; Pme1, molecules with monomethylated proline and/or molecules with dimethylated proline and one acetylated lysine; Pme2, dimethylated
proline; Ac, one acetylated lysine; Pme1 Ac, monomethylated proline and one acetylated lysine and/or peptides with dimethylated proline and two
Nucleic Acids Research,2012, Vol.40, No. 4 1543
methylates H3 but is also able to methylate H2A and H4
albeit to a much lower level. The only methylation site
we could detect on H3 was a mono and dimethylation
of R2, which prevented a cleavage of trypsin at H3R2
leading to a fragment carrying amino acids 1–8. The frag-
mentation spectra also showed that dART8, like its mam-
malian counterpart (49), catalyzes the formation of an
As a knock down of dART8 leads to an increase of H2B
methylation, we wondered whether an increased amount
of dART8 would in turn lead to decreased N-terminal
H2B methylation. We therefore expressed dART8 in
SL2 cells under an inducible promoter and investigated
the degree of H2B methylation after the induction of ex-
pression (Figure 6B). Consistent with a role of dART8 in
the regulation of H2B methylation we detected a strong
reduction of H2B methylation in cells where dART8 ex-
pression had been induced. As the human dART8
ortholog PRMT6 has been shown to act as a transcrip-
tional repressor, we tested whether the reduction of
dART8 leads to an increased expression of dNTMT.
However, we did not detect significant changes of
dNTMT levels dependent on the amount of dART8
(Figure 6C and Supplementary Figure S3). We next
wanted to test whether dART8 might in fact methylate
dNTMT thereby inhibiting its activity. However, we
could neither observe an effect of dART8 on the ability
of dNTMT to methylate H2B when the three proteins
were incubated together in an in vitro methyltransferase
assay nor did we detect a methylation of dNTMT by
dART8 or vice versa (Supplementary Figure S4).
Since both dNTMT and dART8 localize to the nucleus
(Figures 5A and 6D), we wanted to test whether they
physically interact with each other. To do this we ex-
pressed the two recombinant proteins in Escherichia coli
carrying two different tags, mixed them and purified the
complex using either immobilized glutathion or an anti-
FLAG resin (Figure 6E) To test whether the two pro-
teins also interact in vivo, we expressed flag tagged
dART in SL2 cells and purified it using a monoclonal
antibody specific for dNTMT. This interaction was
verified using the reciprocal purification of dNTMT
using an anti-FLAG resin (Figure 6F). Based on the
observed interactions, we assume that the negative regu-
lation of H2B methylation by dART8 is likely to be due to
its interaction with the enzyme responsible for this
Table 2. Predicted targets of dNTMT
Accession numberProtein nameN-terminal sequence N-terminus conserved
Ribosomal protein L12
Testis specific Tetratricopeptid containing protein
Testis-specific ankyrin repeat protein
Sticky protein kinase
Ribosomal protein L23a
SPATA5 spermatogenesis-associated factor
Peptidyl-prolyl cis-trans isomerase
Putative membrane protein
Zn-finger Protein CG8635
OBG like ATPase1
Ribosomal protein S25
Growth arrest-specific 8
BMP receptor IA
CG15890 solute carrier protein
CG42826 solute carrier protein
All proteins that carry a (M)-A/P/S-P-K recognition site on their N-terminus within the predicted proteome of D. melanogaster are listed (67).
1544Nucleic Acids Research, 2012,Vol.40, No. 4
Post-translational modifications in histones play a major
role in setting up specific chromatin structures. In the last
years it became evident that histone modifications can
strongly affect each other thereby generating complex
modification patterns. Several evidences for a crosstalk
among different tails have been reported, suggesting a
complex network and an interdependency of modifications
and the accompanying enzymes. While studying the post-
translational modifications of H2B in D. melanogaster we
found the methylation of its N-terminus as the major
modification of this histone. In contrast to many other
known N-terminal modifications, the methylation is
highly regulated. We identified the enzyme responsible
for establishing this modification (dNTMT/CG1675)
and confirmed that it physically interacts with another
histone methyltransferase (dART8) specific for H3R2
thereby constituting a bifunctional methyltransferase
complex. The two enzymes not only interact with each
other physically but also have opposing functions as the
reduction of dART8 protein levels results in an increase
of N-terminal methylation and the overexpression in a
strong decrease. This effect of the modulation of dART8
concentration on H2B methylation may be due to a
negative crosstalk between the methylation of H3R2
and the N-terminal methylation of H2B or due to a com-
N-terminal methylation is a rare modification in eu-
karyotic proteins and has only recently been addressed
functionally. The methylation of the N-terminus of
human RCC1 has been shown to be important for its
stable interaction with chromatin (38) and its disturbance
leads to mitotic defects in vivo (38). This regulation is in
accordance with the structure of the nucleosome bound
RCC1 molecule, where a N-terminal loop has been sug-
gested to interact with the nucleosomal DNA (50).
Interestingly, despite the strong conservation of RCC1
function in metazoans, the N-terminal methylation site
of RCC1 is not conserved in the Drosophila ortholog of
RCC1, suggesting that the stabilization of RCC1 on chro-
matin is either accomplished by a different mechanism or
not required in Drosophila.
3500 3552 3604 36563708 3760
Figure 5. dNTMT is a nuclear protein and generates highly methylated chromatin in the nucleus. (A) Immunolocalization of FLAG-tagged dNTMT
in Drosophila L2-4 cells transiently transfected with the FMO4789 dNTMT expression plasmid. DNA was stained with ToPro-3 to visualize the
nuclear volume. (B) Isolation of chromatin using micrococcal nuclease (MNase). Chromatin fragments were released from isolated nuclei using
MNase and the DNA was analyzed by agarose gel electrophoresis (left panel) or SDS–PAGE followed by a Coomassie staining (right panel),
respectively (C) MALDI-TOF spectra showing the peptide 1–28 of H2B after acylation and trypsin digestion. The top spectrum is derived from acid
extracted H2B whereas the bottom one is a result of the analysis of chromatin bound H2B. ‘un’=unmodified proline, ‘Pme1’=monomethylated
proline, ‘Pme2’=dimethylated proline.
Nucleic Acids Research,2012, Vol.40, No. 41545
Relative intensity (%)
P met1P met2
dH2B + dART8
dH2B + dART8
3500 3552 36v04
Figure 6. Drosophila ART8 interacts with dNTMT and regulates H2B N-terminal methylation. (A) H2B methylation measured by MALDI-TOF
MS after knockdown of different dART enzymes. The relative intensities of the signals assigned to unmodified (white bars), mono- (gray bars) and
dimethylated proline of H2B were determined after a knockdown of the corresponding enzyme. (B) MALDI-TOF spectra showing the peptide 1–28
of H2B after acylation and trypsin digestion. The spectra are derived from acid extracted H2B from untransfected cells (top), or cells transfected with
an inducible dART8 expression construct before (middle) or after (bottom) induction. un, unmodified proline; Pme1, monomethylated proline; Pme2,
dimethylated proline. (C) Western blot of a whole cell extract after knockdown with dsRNAi against GST (Ctrl.), dART8 (dART8) and dNTMT
(dNTMT). dNTMT was detected by a monoclonal antibody against dNTMT. (D) Immunolocalization of FLAG-HA tagged dART8 in Drosophila
1546Nucleic Acids Research, 2012,Vol.40, No. 4
Eight proteins out of 36 that carry a recognition site for
a presumptive N-terminal methyltransferases are con-
served between humans and fruit flies. Those are three
ribosomal subunits, two proteins associated with stress
response and/or growth arrest, two with an unknown
function and one member of the respiratory chain.
Considering the conservation of the enzyme responsible
for this modification, the low degree of overlap is very
surprising. However, it is striking that frequently the
human orthologous of proteins that carry a recognition
site only in Drosophila can be found associated with
putative targets in humans. So is for example RCC1
(methylated in humans but not in Drosophila) in a
complex with H2B (methylated in Drosophila but not in
humans) (50) and many factors that are predicted to
become N-terminally methylated are expressed in a testis
specific manner in humans as well as in Drosophila
[compare (39) to Table 2]. This suggests that the
N-terminal methylation exerts its function on a protein
complex as long as one subunit carries the modification.
What may be the function of H2B methylation in
Drosophila? The observation that it increases during tem-
perature stress as well as during differentiation points
towards a role of H2B methylation in stabilizing chroma-
tin. In both circumstances (heat shock and aging) the
overall transcription is reduced and becomes restricted
to a limited number of active genes (51,52). At the same
time, we detect only low levels of methylation at early
stages of embryonic development, where chromatin has
been reported to be hyper-dynamic in other systems
(53). In general, histones have been shown to have a
higher turnover in dynamically transcribed regions com-
pared to non-transcribed domains (54) therefore lowering
this turnover may have a repressive effect on general tran-
scriptional activity. As N-terminal modifications have
been shown to regulate protein turnover (55), H2B methy-
lation might similarly stabilize the protein and contribute
to a reduced overall transcription in differentiated or
stressed cells. Alternatively, H2B methylation could also
be a consequence of low transcriptional activity, which is
the supported by the observation that it also increases
when cells are treated with transcriptional inhibitors or
with inhibitors of TopoII (35,56).
N-terminal modification of histones is not restricted to
H2B in flies but has also been detected in H4, which is
N-acetylated in virtually all eukaryotes (57–59), H2A,
which is N-acetylated in human tissue culture cells (60)
and H2B from yeast, which is also acetylated at its
N-terminal residue (61). None of the N-acetylations
have been associated with a particular function and are
thought to be constitutive modifications following histone
synthesis. Our description of a developmentally and
stress-induced regulated N-terminal modification of H2B
sheds new light on the potential function of this modifica-
tion in chromatin metabolism.
The relative expression levels of dNTMT during differ-
ent developmental stages correlate very well with the rela-
tive levels of H2B methylation [compare Figure 2 with
Supplementary Figure S5 (62)]. This suggests that the pro-
portion of H2B methylation is regulated by the amount of
enzyme present in the cell. However, in SL2 cells we also
observe that the level of dART8 regulates H2B methyla-
tion, which suggests a second layer of control for H2B
methylation. Although the two proteins interact physical-
ly, they do not methylate each other and the interaction
does not lead to an alteration of dNTMTs activity. The
observed interference of dART8 expression with H2B
methylation can therefore not be explained by a direct
effect mediated by the simple interaction of the two poly-
peptides. Expression studies show that the dNTMT ex-
pression is not downregulated by dART8 expression or
upregulated in cells that lack dART8.
Recently local SAM concentrations within the nucleus
have been suggested to play an important role in
regulating the activity of histone methyltransferases (63).
The regulation of H2B methylation we observe in vivo by
modulating dART8 levels may therefore be due to a com-
petition of the two enzymes residing in the same complex
for the limiting cofactor SAM. Several nuclear complexes
have been shown to contain multiple methyltransferases
activities (64–66) that could potential be regulated by a
similar mechanism. Alternatively, as dART8 methylates
R2 at H3, a crosstalk between the two histone-tails
where methylation of H3R2 inhibits H2B N-terminal
methylation could also be an explanation for the recipro-
cal activities of the two enzymes. Based on our data,
future studies are necessary that distinguish the pos-
sible mechanisms of how dART8 modulates dNTMTs
activity within a single protein complex and analyze the
role of the striking increase in H2Bmethylation during fly
Supplementary Data are
Supplementary Figures 1–5, Supplementary Reference .
We are grateful to P. Schilcher for expert technical assist-
ance, to Teresa Barth for help with the FACS measure-
ments and to all members of the Imhof groups for critical
reading of the manuscript and helpful comments.
Figure 6. Continued
SL2 cells transfected with an inducible FHdART8 expression plasmid. DNA was stained with DAPI to visualize the nuclear volume. (E) In vitro
interaction of flag-dNMT and GST–dART8. Recombinant proteins were mixed and incubated for 10min at room temperature the complexes were
subsequently either purified by glutathione-(GST) or M2-(FLAG) agarose., separated via SDS–PAGE, blotted and detected using either an anti-GST
(top panel) or an anti-FLAG (bottom panel) antibody. (F) Interaction of dART8 and dNTMT. Whole cell extracts from cells expressing Flag tagged
dART8 were either purified by M2 (FLAG) agarose, an immobilized monoclonal dNTMT antibody (dNTMT) or a non-related immobilized
antibody (Ctrl.). 1 and 5% of Input as well as the immunoprecipitated material were separated by SDS–PAGE, blotted and detected by either
an anti-FLAG (top panel) or an anti-dNTMT antibody (bottom panel).
Nucleic Acids Research,2012, Vol.40, No. 41547
Funding for open access charge: European Union (LSHG-
Conflict of interest statement. None declared.
1. Bhaumik,S.R., Smith,E. and Shilatifard,A. (2007) Covalent
modifications of histones during development and disease
pathogenesis. Nat. Struct. Mol. Biol., 14, 1008–1016.
2. Bannister,A.J. and Kouzarides,T. (2011) Regulation of chromatin
by histone modifications. Cell Res., 21, 381–395.
3. Kouzarides,T. (2007) Chromatin modifications and their function.
Cell, 128, 693–705.
4. Wang,G.G., Allis,C.D. and Chi,P. (2007) Chromatin remodeling
and cancer, Part I: covalent histone modifications.
Trends Mol. Med., 13, 363–372.
5. Jenuwein,T. and Allis,C.D. (2001) Translating the histone code.
Science, 293, 1074–1080.
6. Turner,B.M. (2008) Simplifying a complex code. Nat. Struct.
Mol. Biol., 15, 542–544.
7. Schotta,G., Lachner,M., Peters,A.H. and Jenuwein,T. (2004) The
indexing potential of histone lysine methylation. Novartis Found
Symp., 259, 22–37; discussion 37–47, 163–169.
8. Wang,Z., Zang,C., Rosenfeld,J.A., Schones,D.E., Barski,A.,
Cuddapah,S., Cui,K., Roh,T.Y., Peng,W., Zhang,M.Q. et al.
(2008) Combinatorial patterns of histone acetylations and
methylations in the human genome. Nat. Genet., 40, 897–903.
9. Barski,A., Cuddapah,S., Cui,K., Roh,T.Y., Schones,D.E.,
Wang,Z., Wei,G., Chepelev,I. and Zhao,K. (2007) High-resolution
profiling of histone methylations in the human genome. Cell, 129,
10. Alvarez,F., Munoz,F., Schilcher,P., Imhof,A., Almouzni,G. and
Loyola,A. (2011) Sequential establishment of marks on soluble
histones h3 and h4. J. Biol. Chem., 286, 17714–17721.
11. Jasencakova,Z., Scharf,A.N., Ask,K., Corpet,A., Imhof,A.,
Almouzni,G. and Groth,A. (2010) Replication stress interferes
with histone recycling and predeposition marking of new histones.
Mol. Cell, 37, 736–743.
12. Loyola,A., Tagami,H., Bonaldi,T., Roche,D., Quivy,J.P.,
Imhof,A., Nakatani,Y., Dent,S.Y. and Almouzni,G. (2009) The
HP1alpha-CAF1-SetDB1-containing complex provides H3K9me1
for Suv39-mediated K9me3 in pericentric heterochromatin.
EMBO Rep., 10, 769–775.
13. Loyola,A., Bonaldi,T., Roche,D., Imhof,A. and Almouzni,G.
(2006) PTMs on H3 variants before chromatin assembly
potentiate their final epigenetic state. Mol. Cell, 24, 309–316.
14. Campos,E.I., Fillingham,J., Li,G., Zheng,H., Voigt,P., Kuo,W.H.,
Seepany,H., Gao,Z., Day,L.A., Greenblatt,J.F. et al. (2010) The
program for processing newly synthesized histones H3.1 and H4.
Nat. Struct. Mol. Biol., 17, 1343–1351.
15. Taverna,S.D., Li,H., Ruthenburg,A.J., Allis,C.D. and Patel,D.J.
(2007) How chromatin-binding modules interpret histone
modifications: lessons from professional pocket pickers.
Nat. Struct. Mol. Biol., 14, 1025–1040.
16. Maurer-Stroh,S., Dickens,N.J., Hughes-Davies,L., Kouzarides,T.,
Eisenhaber,F. and Ponting,C.P. (2003) The Tudor domain ‘Royal
Family’: Tudor, plant Agenet, Chromo, PWWP and MBT
domains. Trends Biochem Sci., 28, 69–74.
17. Lo,W.S., Duggan,L., Emre,N.C., Belotserkovskya,R., Lane,W.S.,
Shiekhattar,R. and Berger,S.L. (2001) Snf1—a histone kinase that
works in concert with the histone acetyltransferase Gcn5 to
regulate transcription. Science, 293, 1142–1146.
18. Clayton,A.L., Rose,S., Barratt,M.J. and Mahadevan,L.C. (2000)
Phosphoacetylation of histone H3 on c-fos- and c-jun-associated
nucleosomes upon gene activation. EMBO J., 19, 3714–3726.
19. Hyllus,D., Stein,C., Schnabel,K., Schiltz,E., Imhof,A., Dou,Y.,
Hsieh,J. and Bauer,U.M. (2007) PRMT6-mediated methylation of
R2 in histone H3 antagonizes H3 K4 trimethylation. Genes Dev.,
20. Kirmizis,A., Santos-Rosa,H., Penkett,C.J., Singer,M.A.,
Vermeulen,M., Mann,M., Bahler,J., Green,R.D. and
Kouzarides,T. (2007) Arginine methylation at histone H3R2
controls deposition of H3K4 trimethylation. Nature, 449,
21. Metzger,E., Imhof,A., Patel,D., Kahl,P., Hoffmeyer,K.,
Friedrichs,N., Muller,J.M., Greschik,H., Kirfel,J., Ji,S. et al.
(2010) Phosphorylation of histone H3T6 by PKCbeta(I) controls
demethylation at histone H3K4. Nature, 464, 792–796.
22. Lee,J.S., Shukla,A., Schneider,J., Swanson,S.K., Washburn,M.P.,
Florens,L., Bhaumik,S.R. and Shilatifard,A. (2007) Histone
crosstalk between H2B monoubiquitination and H3 methylation
mediated by COMPASS. Cell, 131, 1084–1096.
23. Briggs,S.D., Xiao,T., Sun,Z.W., Caldwell,J.A., Shabanowitz,J.,
Hunt,D.F., Allis,C.D. and Strahl,B.D. (2002) Gene silencing:
trans-histone regulatory pathway in chromatin. Nature, 418, 498.
24. Zippo,A., Serafini,R., Rocchigiani,M., Pennacchini,S.,
Krepelova,A. and Oliviero,S. (2009) Histone crosstalk between
H3S10ph and H4K16ac generates a histone code that mediates
transcription elongation. Cell, 138, 1122–1136.
25. Wang,H., Wang,L., Erdjument-Bromage,H., Vidal,M., Tempst,P.,
Jones,R.S. and Zhang,Y. (2004) Role of histone H2A
ubiquitination in Polycomb silencing. Nature, 431, 873–878.
26. Nakanishi,S., Lee,J.S., Gardner,K.E., Gardner,J.M.,
Takahashi,Y.H., Chandrasekharan,M.B., Sun,Z.W., Osley,M.A.,
Strahl,B.D., Jaspersen,S.L. et al. (2009) Histone H2BK123
monoubiquitination is the critical determinant for H3K4 and
H3K79 trimethylation by COMPASS and Dot1. J. Cell Biol.,
27. Cheung,W.L., Ajiro,K., Samejima,K., Kloc,M., Cheung,P.,
Mizzen,C.A., Beeser,A., Etkin,L.D., Chernoff,J., Earnshaw,W.C.
et al. (2003) Apoptotic phosphorylation of histone H2B is
mediated by mammalian sterile twenty kinase. Cell, 113, 507–517.
28. Ajiro,K., Scoltock,A.B., Smith,L.K., Ashasima,M. and
Cidlowski,J.A. (2010) Reciprocal epigenetic modification of
histone H2B occurs in chromatin during apoptosis in vitro and
in vivo. Cell Death Differ., 17, 984–993.
29. Bonenfant,D., Towbin,H., Coulot,M., Schindler,P., Mueller,D.R.
and van Oostrum,J. (2007) Analysis of dynamic changes in
post-translational modifications of human histones during cell
cycle by mass spectrometry. Mol. Cell Proteomics, 6, 1917–1932.
30. Prentice,D.A., Loechel,S.C. and Kitos,P.A. (1982) Histone H2A
phosphorylation in animal cells: functional considerations.
Biochemistry, 21, 2412–2420.
31. Bonenfant,D., Coulot,M., Towbin,H., Schindler,P. and van
Oostrum,J. (2006) Characterization of histone H2A and H2B
variants and their post-translational modifications by mass
spectrometry. Mol. Cell Proteomics, 5, 541–552.
32. Weake,V.M., Lee,K.K., Guelman,S., Lin,C.H., Seidel,C.,
Abmayr,S.M. and Workman,J.L. (2008) SAGA-mediated H2B
deubiquitination controls the development of neuronal
connectivity in the Drosophila visual system. EMBO J., 27,
33. Bray,S., Musisi,H. and Bienz,M. (2005) Bre1 is required for
Notch signaling and histone modification. Dev. Cell., 8, 279–286.
34. Maile,T., Kwoczynski,S., Katzenberger,R.J., Wassarman,D.A. and
Sauer,F. (2004) TAF1 activates transcription by phosphorylation
of serine 33 in histone H2B. Science, 304, 1010–1014.
35. Desrosiers,R. and Tanguay,R.M. (1988) Methylation of
Drosophila histones at proline, lysine, and arginine residues
during heat shock. J. Biol. Chem., 263, 4686–4692.
36. Webb,K.J., Lipson,R.S., Al-Hadid,Q., Whitelegge,J.P. and
Clarke,S.G. (2010) Identification of protein N-terminal
methyltransferases in yeast and humans. Biochemistry, 49,
37. Camato,R. and Tanguay,R.M. (1982) Changes in the methylation
pattern of core histones during heat-shock in Drosophila cells.
EMBO J., 1, 1529–1532.
38. Chen,T., Muratore,T.L., Schaner-Tooley,C.E., Shabanowitz,J.,
Hunt,D.F. and Macara,I.G. (2007) N-terminal alpha-methylation
of RCC1 is necessary for stable chromatin association and
normal mitosis. Nat. Cell Biol., 9, 596–603.
1548 Nucleic Acids Research, 2012,Vol.40, No. 4
39. Tooley,C.E., Petkowski,J.J., Muratore-Schroeder,T.L., Download full-text
Balsbaugh,J.L., Shabanowitz,J., Sabat,M., Minor,W., Hunt,D.F.
and Macara,I.G. (2010) NRMT is an alpha-N-methyltransferase
that methylates RCC1 and retinoblastoma protein. Nature, 466,
40. Bonaldi,T., Imhof,A. and Regula,J.T. (2004) A combination of
different mass spectroscopic techniques for the analysis of
dynamic changes of histone modifications. Proteomics, 4,
41. Villar-Garea,A. and Imhof,A. (2008) Fine mapping of
posttranslational modifications of the linker histone H1 from
Drosophila melanogaster. PLoS ONE, 3, e1553.
42. Villar-Garea,A., Israel,L. and Imhof,A. (2008) Analysis of histone
modifications by mass spectrometry. Curr. Protoc. Protein Sci.,
Chapter 14, Unit 14 10.
43. Olsen,J.V., de Godoy,L.M., Li,G., Macek,B., Mortensen,P.,
Pesch,R., Makarov,A., Lange,O., Horning,S. and Mann,M. (2005)
Parts per million mass accuracy on an Orbitrap mass
spectrometer via lock mass injection into a C-trap. Mol. Cell.
Proteomics, 4, 2010–2021.
44. Bunch,T.A., Grinblat,Y. and Goldstein,L.S. (1988)
Characterization and use of the Drosophila metallothionein
promoter in cultured Drosophila melanogaster cells.
Nucleic Acids Res., 16, 1043–1061.
45. Schneider,I. (1972) Cell lines derived from late embryonic stages
of Drosophila melanogaster. J. Embryol. Exp. Morphol., 27,
46. Echalier,G. and Ohanessian,A. (1969) [Isolation, in tissue culture,
of Drosophila melangaster cell lines]. C. R. Acad. Sci. Hebd.
Seances Acad. Sci. D, 268, 1771–1773.
47. Wullschleger,S., Loewith,R. and Hall,M.N. (2006) TOR signaling
in growth and metabolism. Cell, 124, 471–484.
48. Giot,L., Bader,J.S., Brouwer,C., Chaudhuri,A., Kuang,B., Li,Y.,
Hao,Y.L., Ooi,C.E., Godwin,B., Vitols,E. et al. (2003) A protein
interaction map of Drosophila melanogaster. Science, 302,
49. Brame,C.J., Moran,M.F. and McBroom-Cerajewski,L.D. (2004) A
mass spectrometry based method for distinguishing between
symmetrically and asymmetrically dimethylated arginine residues.
Rapid Commun. Mass. Spectrom., 18, 877–881.
50. Makde,R.D., England,J.R., Yennawar,H.P. and Tan,S. (2010)
Structure of RCC1 chromatin factor bound to the nucleosome
core particle. Nature, 467, 562–566.
51. Vazquez,J., Pauli,D. and Tissieres,A. (1993) Transcriptional
regulation in Drosophila during heat shock: a nuclear run-on
analysis. Chromosoma, 102, 233–248.
52. Lu,B.Y., Ma,J. and Eissenberg,J.C. (1998) Developmental
regulation of heterochromatin-mediated gene silencing in
Drosophila. Development, 125, 2223–2234.
53. Meshorer,E., Yellajoshula,D., George,E., Scambler,P.J.,
Brown,D.T. and Misteli,T. (2006) Hyperdynamic plasticity of
chromatin proteins in pluripotent embryonic stem cells. Dev. Cell,
54. Deal,R.B., Henikoff,J.G. and Henikoff,S. (2010) Genome-wide
kinetics of nucleosome turnover determined by metabolic labeling
of histones. Science, 328, 1161–1164.
55. Van Damme,P., Arnesen,T. and Gevaert,K. (2011) Protein
alpha-N-acetylation studied by N-terminomics. FEBS J., 278,
56. Desrosiers,R. and Tanguay,R.M. (1986) Further characterization
of the posttranslational modifications of core histones in response
to heat and arsenite stress in Drosophila. Biochem. Cell Biol., 64,
57. Bonaldi,T., Regula,J.T. and Imhof,A. (2004) The use of mass
spectrometry for the analysis of histone modifications.
Methods Enzymol., 377, 111–130.
58. Ruiz-Carrillo,A., Wangh,L.J. and Allfrey,V.G. (1975)
Processing of newly synthesized histone molecules. Science, 190,
59. Pesavento,J.J., Bullock,C.R., LeDuc,R.D., Mizzen,C.A. and
Kelleher,N.L. (2008) Combinatorial modification of human
histone H4 quantitated by two-dimensional liquid
chromatography coupled with top down mass spectrometry.
J. Biol. Chem., 283, 14927–14937.
60. Boyne,M.T. 2nd, Pesavento,J.J., Mizzen,C.A. and Kelleher,N.L.
(2006) Precise characterization of human histones in the H2A
gene family by top down mass spectrometry. J. Proteome Res., 5,
61. Mullen,J.R., Kayne,P.S., Moerschell,R.P., Tsunasawa,S.,
Gribskov,M., Colavito-Shepanski,M., Grunstein,M., Sherman,F.
and Sternglanz,R. (1989) Identification and characterization of
genes and mutants for an N-terminal acetyltransferase from yeast.
EMBO J., 8, 2067–2075.
62. Celniker,S.E., Dillon,L.A., Gerstein,M.B., Gunsalus,K.C.,
Henikoff,S., Karpen,G.H., Kellis,M., Lai,E.C., Lieb,J.D.,
MacAlpine,D.M. et al. (2009) Unlocking the secrets of the
genome. Nature, 459, 927–930.
63. Katoh,Y., Ikura,T., Hoshikawa,Y., Tashiro,S., Ito,T., Ohta,M.,
Kera,Y., Noda,T. and Igarashi,K. (2011) Methionine
adenosyltransferase II serves as a transcriptional corepressor of
Maf oncoprotein. Mol. Cell., 41, 554–566.
64. Fritsch,L., Robin,P., Mathieu,J.R., Souidi,M., Hinaux,H.,
Rougeulle,C., Harel-Bellan,A., Ameyar-Zazoua,M. and
Ait-Si-Ali,S. (2010) A subset of the histone H3 lysine 9
methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate
in a multimeric complex. Mol. Cell, 37, 46–56.
65. Tachibana,M., Ueda,J., Fukuda,M., Takeda,N., Ohta,T.,
Iwanari,H., Sakihama,T., Kodama,T., Hamakubo,T. and
Shinkai,Y. (2005) Histone methyltransferases G9a and GLP form
heteromeric complexes and are both crucial for methylation of
euchromatin at H3-K9. Genes Dev., 19, 815–826.
66. Esteve,P.O., Chin,H.G., Smallwood,A., Feehery,G.R.,
Gangisetty,O., Karpf,A.R., Carey,M.F. and Pradhan,S. (2006)
Direct interaction between DNMT1 and G9a coordinates DNA
and histone methylation during replication. Genes Dev., 20,
67. Tweedie,S., Ashburner,M., Falls,K., Leyland,P., McQuilton,P.,
Marygold,S., Millburn,G., Osumi-Sutherland,D., Schroeder,A.,
Seal,R. et al. (2009) FlyBase: enhancing Drosophila Gene
Ontology annotations. Nucleic Acids Res., 37, D555–559.
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