Download In vivo assays to study histone ubiquitylation

Methods 31 (2003) 59–66
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In vivo assays to study histone ubiquitylation
Cheng-Fu Kao and Mary Ann Osley*
Molecular Genetics and Microbiology, University of New Mexico Health Science Center, 915 Camino de Salud, Albuquerque, NM 87131, USA
Accepted 19 February 2003
Abstract
The importance of histone acetylation, phosphorylation, and methylation in transcription and other DNA-mediated processes is
now well established. Histones are also ubiquitylated, but in contrast to the majority of ubiquitylated proteins, ubiquitylated histones are not generally targeted for degradation and may play roles similar to those of other histone modifications. Antibodies
against acetylated histones have provided unique insights into the regulation, distribution, and cellular roles of these modified
histones. In this report, we describe methods to identify ubiquitylated histones in budding yeast and HeLa cells. We provide
protocols to detect ubiquitylated histones that are based on a combination of in vivo genetic and immunological assays. These
methods should provide relatively simple and useful tools to study the global regulation of this important but poorly understood
histone modification.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Histones; Ubiquitylation; In vivo detection assays
1. Introduction
Once thought to provide merely a structural role in
compacting chromatin, nucleosomes are now recognized
as important players in all processes that occur on eukaryotic chromosomes [1,2]. Chromatin is in fact highly
dynamic, undergoing global changes in folding during
mitosis and local changes in folding during gene expression. Posttranslational modifications of histones,
the protein constituents of nucleosomes, contribute to
the dynamic nature of chromatin [3,4]. These modifications, which include acetylation, phosphorylation, and
methylation, are generally targeted to the flexible Nterminal tails of the four core histones, where their
presence leads to chromatin states that are either ‘‘open’’
or ‘‘closed’’ with respect to transcriptional activity [5–7].
The histones H2A, H2B, H3, H2A.Z, and H1 are also
modified by ubiquitylation, in which the 76-amino-acid
protein ubiquitin is attached via an isopeptide linkage
between its terminal glycine residue and an e amino
group of lysine in the histone protein [8–15]. Acceptor
lysines have been identified in the C-terminal tails of
*
Corresponding author. Fax: 1-505-272-6029.
E-mail address: [email protected] (M.A. Osley).
histones H2A (lysine 119) and H2B (lysine 120 in vertebrates and lysine 123 of yeast), but the sites of
ubiquitin attachment in other histones remain unknown
[13,16–18]. Ubiquitylated histones are found in a wide
range of organisms and cell types, with estimates of their
abundance ranging from 1% to 15% of unmodified
histones [8,11,13,14,19]. Like acetylation, ubiquitylation
is a highly dynamic and reversible histone modification.
In cycling vertebrate cells ubiquitylated histones have
been found to turn over rapidly, and during mitosis
there is a global cycle of histone deubiquitylation
[11,12,20–24]. Deubiquitylation may be accomplished
by a family of ubiquitin-specific proteases or Ubps,
some of which may target histones [25–27].
Although protein ubiquitylation is a widely used
mechanism to target proteins for degradation, histone
ubiquitylation appears to play a different role [26,28–30].
Histones are predominantly monoubiquitylated in vivo,
a modification that, unlike polyubiquitylation, is not
associated with protein turnover. Moreover, ubiquitylated histones have been identified as stable constituents
of nucleosomes in chromatin isolated from both fly and
vertebrate cells [31–34]. The effect of histone ubiquitylation on chromatin structure is largely unknown
[35]. Mononucleosomes reconstituted in vitro from
1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S1046-2023(03)00088-4
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C.-F. Kao, M.A. Osley / Methods 31 (2003) 59–66
ubiquitylated H2A or H2B (uH2A or uH2B) appear
similar in many parameters to nucleosomes containing
unmodified histones [36]. Because ubiquitin is a bulky
moiety, it has been postulated that its attachment to
histones could disrupt chromatin folding [35,37]. This
argument tends to be supported by the observation that
metazoan histones are globally deubiquitylated at metaphase, when chromatin becomes highly compacted
[11,21]. However, in the absence of linker histones, nucleosome arrays reconstituted in vitro from ubiquitylated
H2A show degrees of folding similar to those of arrays
formed from unmodified H2A [38]. Thus, histone ubiquitylation could form part of the so-called ‘‘histone code,’’
acting as a tag or recognition element to direct proteins
such as chromatin remodeling factors, transcription factors, and repair and replication factors to specific chromosomal domains [39]. The notion that ubiquitylated
histones serve as a mark to direct the localization of specific factors to chromatin is supported by recent studies
showing that yeast uH2B has differential effects on the
methylation of adjacent lysine residues in the H3 N tail by
several SET domain proteins [40–43]. These results are
not consistent with a model in which histone ubiquitylation merely serves a structural role to open chromatin.
Ubiquitylation of proteins destined for degradation
occurs through a concerted series of enzymatic reactions
initiated by a ubiquitin-activating enzyme [28–30]. Activated ubiquitin is then attached via a thiolester linkage
to a cysteine residue in one of many different ubiquitinconjugating enzymes (Ubcs) or E2s. Ubiquitin is finally
transferred to a lysine residue in target proteins through
the activity of a family of ubiquitin ligases or E3s, which
have either catalytic or regulatory roles. These latter
factors confer specificity by targeting ubiquitin attachment to particular substrates. Until recently, the factors
involved in histone ubiquitylation were unknown. The
most likely candidate for a histone-specific Ubc was
Rad6, an evolutionarily conserved ubiquitin-conjugating enzyme that is able to ubiquitylate free histones in
vitro [44–48]. Rad6 was shown to be required for monoubiquitylation of yeast H2B in vivo, although it is not
known if it performs a similar role in other eukaryotic
cells [13,49]. In vitro, Rad6 ubiquitylates histones in the
absence of an E3 [44,45,48]. Three E3s that interact directly or indirectly with Rad6 (Rad18, Rad5, and Ubr1)
in vivo are dispensable for H2B ubiquitylation in yeast
[41]. While this suggests that Rad6 ubiquitylates cellular
H2B in the absence of an E3, the dynamic changes in the
levels of ubiquitylated histones during transcription and
other cellular processes argues for the existence of
specificity factors [8,13,33,49–52]. Support for this view
comes from two recent reports that have identified the
yeast Bre1 protein as an E3 that appears to target Rad6
to H2B at a constitutively transcribed gene [53,54].
The biological roles of ubiquitylated histones appear
to be wide and varied. Both uH2A and uH2B have been
localized to nucleosomes that flank transcriptionally
active genes [31,33,50]. This has led to the hypothesis
that histone ubiquitylation might be a prerequisite for
activated transcription, although transcription itself
might also create a chromatin environment that is permissive for ubiquitylation [55,56]. Recent studies have
also implicated uH2B in both gene repression and heterochromatic gene silencing [40–43,57]. uH2A, uH2B,
and uH3 have each been connected to meiosis, and
uH2A has been shown to be concentrated in replication
foci in some transformed cell types [8,13,49,52]. In addition, the global cycle of histone deubiquitylation/ubiquitylation that occurs during metazoan mitoses
implicates this histone modification in aspects of chromosome segregation [11]. Finally, yeast H2B has shown
to have a regulatory role with respect to the methylation
of specific lysine residues in histone H3 [40]. However, it
is important to point out that in no case is it known how
ubiquitylated histones function. As discussed above, this
function could be structural or regulatory, or some
combination of both mechanisms.
For researchers interested in histone modifications,
there are a number of questions relating to the presence
of ubiquitylated histones in eukaryotic cells. Which histones are ubiquitylated? How abundant are these
modified histones? How is histone ubiquitylation regulated and distributed in vivo? What are the biological
roles of ubiquitylated histones? Key to answering many
of these questions is a sensitive method to detect ubiquitylated histones in the cell. Antibodies against specific
acetylated lysine residues in histones have been instrumental in unraveling the regulation and biological roles
of these particular histone modifications. However, only
a few antibodies have been described that specifically
recognize ubiquitylated histones and only antibodies
against ubiquitylated H2A have become commercially
available [52]. Nonetheless, detection of ubiquitylated
histones is possible using antibodies that recognize free
ubiquitin and ubiquitin–protein conjugates, as well as
through genetic approaches that use epitope-tagged histones and ubiquitin. In the following section, several
methods are outlined to detect ubiquitylated histones in
vivo in two eukaryotic organisms. Emphasis is placed on
detection of these modified histones in the budding
yeast, Saccharomyces cerevisiae, where genetic approaches have provided sensitive assays for the regulation and biological roles of ubiquitylated histones [13].
2. Methods
2.1. Identification of ubiquitylated histones in budding
yeast
The low histone gene copy number in yeast, coupled
with the genetic tractability of this organism, has made
C.-F. Kao, M.A. Osley / Methods 31 (2003) 59–66
it possible to replace chromosomal histone genes with
epitope-tagged versions of these genes. Yeast strains
have been constructed that contain a single H2B gene
with a Flag epitope at its amino terminus, which does
not appear to interfere with the function of H2B in cell
growth or other measurable in vivo processes [56]. The
Flag epitope provides a convenient immunological tag
to identify H2B and its modified forms by Western blot
analysis, and histone H2B that carries the Flag tag can
be quantitatively immunoprecipitated from yeast cell
lysates [13,58]. The methods outlined below with strains
containing Flag-H2B could in principle be applied to
strains containing Flag-tagged versions of the other core
histones or histone variants. Moreover, for many studies, Flag-tagged histones can be examined in the presence of untagged histones (C. Kao and M.A. Osley,
unpublished observations).
2.1.1. Strains and plasmids
Saccharomyces cerevisiae strain JR5-2A (MAT a
htb1-1 htb2-1 ura3-1 leu2-3, -112, his3-11, -15 trp1-1
ade2-1 can1-100 hpRS314-Flag-HTB1 or pRS314-Flaghtb1-K123Ri is used as the starting strain for the
methods described below [13,58]. Plasmid pRS314-FlagHTB1 is a CEN/TRP1 vector that carries the HTB1
gene with a single Flag epitope inserted at the HTB1
ATG codon. Plasmid pRS314-Flag-htb1-K123R carries
the same epitope-tagged HTB1 gene with a mutation
that changes lysine 123 to arginine, thus abolishing the
conserved ubiquitylation site. To provide enhanced detection of ubiquitylated H2B, JR5-2A can be transformed with plasmid p81, a high-copy HIS3 vector that
carries a GAL1-regulated UBI4 gene with the HA epitope inserted at its amino terminus [13].
3.
4.
5.
6.
7.
8.
2.1.2. TCA lysate preparation and imunoprecipitation
Breakage of yeast cells in the presence of TCA preserves ubiquitin–histone conjugates by inactivating isopeptidases. The method described below is adapted from
a protocol described in Ohashi et al. [59], as further
modified by Robzyk [13]. TCA lysates can be used to
examine bulk levels of ubiquitylated histones in yeast
extracts, and they are the starting point for immunoprecipitation of Flag epitope-tagged histones.
1. Fifty milliliters of cells are grown at 30 °C in synthetic dextrose (SD) medium lacking tryptophan to
an OD600 of 0.8 (40 OD units). For strains that contain the GAL-regulated HA-UBI4 gene, cells are
grown to the same OD600 in the presence of 2% galactose instead of 2% dextrose. SD medium contains
6.4 g of yeast nitrogen base minus amino acids (Difco) per liter of sterile distilled water. Amino acids are
supplemented as required, and cells are grown at
30 °C with shaking.
2. Cells are centrifuged at 4500 rpm for 5 min in a tabletop centrifuge (Fisher), and the cell pellet is immedi-
9.
10.
11.
12.
61
ately resuspended in 5–10 ml of 20% TCA (trichloroacetic acid). Cells are again centrifuged at 4500 rpm
for 5 min and all residual TCA is removed from the
cell pellet, which is immediately frozen in an ethanol–dry ice bath and stored at )80 °C.
The cell pellet is thawed on ice, resuspended in 0.2 ml
of 20% TCA, and transferred to a 1.5-ml microfuge
tube. The cells are broken by vortexing at the highest
speed for 2 min at 4 °C with 0.4 g of acid-washed
glass beads (0.2 mm, Thomas).
The broken cell pellet is transferred to a new microfuge tube, avoiding the glass beads, and combined
with two 500-ll washes of the beads with 5% TCA.
The lysate will be in 1.2 ml of TCA at the end of
the washes.
The lysate is incubated on ice for 10 min or longer,
and the precipitated protein is collected by centrifugation at 14,000 rpm for 15 min at 4 °C. The supernatant fraction is aspirated, and the precipitated
proteins are briefly recentrifuged and aspirated to remove as much TCA as possible.
The precipitated proteins are resuspended in 750 ll
of 1 Laemmli sample buffer (0.06 M Tris, pH 6.8,
10% (v/v) SDS, 5% (v/v) fresh 2-mercaptoethanol,
and 0.0025% (w/v) bromphenol blue) and 50 ll of
unbuffered 2 M Tris is added to neutralize the pH
of the lysate. The lysate will turn from a yellow to
a blue color when the pH is neutralized.
The suspension is boiled for 5 min and clarified by
centrifugation at 14,000 rpm for 10 min at room temperature. The clarified lysate is transferred to a fresh
microfuge tube and either directly loaded onto an
SDS–polyacrylamide gel, used for immunoprecipitation, or stored at )80 °C.
The ‘‘concentration’’ of the protein lysate is calculated as OD600 equivalents. Typically, 0.5–1.0 OD
units of the lysate is analyzed in Western blots, while
10–20 OD units is used for immunoprecipitation.
Flag-H2B is immunoprecipitated from lysates by diluting the lysate 1:5 into IP buffer [50 mM Tris, pH
7.4, 150 mM NaCl, 0.5% Nonidet p-40 (NP-40), and
0.5 mg/ml bovine serum albumin (BSA)]. Thirty microliters of anti-Flag M2 agarose beads (Sigma) are
added and incubation is carried out for 1 h at 4 °C.
The beads are collected by centrifugation at
2000 rpm for 2 min and washed one time with IP
buffer containing BSA, and three times with IP buffer without BSA.
Proteins bound to the resin are eluted directly into
50 ll of 1 Laemmli sample buffer by boiling for 5 min.
Ten microliters of the immunoprecipitate, or 0.5–1.0
OD units of cell lysate, are analyzed by electrophoresis on 8.7 8.3-cm 15% acrylamide SDS–polyacrylamide gels (Hoeffer Mighty Small SE260 apparatus)
run in Tris–glycine–SDS buffer (25 mM Tris,
192 mM glycine, 0.1% SDS, pH 8.3) at 150 V for 3 h.
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C.-F. Kao, M.A. Osley / Methods 31 (2003) 59–66
13. Following electrophoresis, proteins are transferred
to an Immobilon-P membrane (Fisher) in Tris–glycine buffer containing 20% methanol at 300 mA for
40 min using an Amersham Mighty Small Transphor
apparatus.
14. Western blot analysis is performed after blocking
the membrane in 50 ml of TBS–Tween (50 mM Tris,
138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH
8.0) containing 5% milk at 4 °C overnight. The
membrane is incubated with primary antibody
against the Flag or HA epitope for 2 h at room
temperature. M2 monoclonal antibody (Sigma) is
diluted 1:300 and anti-HA monoclonal antibody
(HA.11, Covance or 12C5A, Roche) is diluted
1:500 into 15–20 ml of TBS–Tween containing 5%
milk. The membrane is washed three times for
15 min each in 15 ml of TBS–Tween, and then incubated for 1 h with sheep anti-mouse Ig (Amersham)
diluted 1:5,000 in 15 ml of TBS–Tween. After four
15-min washes in 15 ml of TBS–Tween, the membrane is developed using an enhanced chemiluminescence kit from either Bio-Rad or Perkin
Elmer–New England Nuclear, following the manufacturerÕs directions.
Western blots probed with anti-Flag antibodies reveal that on 15% SDS–polyacrylamide gels unmodified
Flag-H2B migrates with an apparent mass of 22 kDa,
while Flag-H2B containing a single ubiquitin conjugate
migrates with a mass of 28 kDa (Fig. 1). If HAubiquitin is expressed, two ubiquitin conjugates are
observed on the anti-Flag blot: one migrating at
28 kDa and a second, representing an HA-ubiquitin
conjugate of Flag-H2B, migrating at 30 kDa (Fig. 2,
left). Blots probed with anti-HA reveal only the 30kDa species (Fig. 2, right). Confirmation that the
slower-migrating species represent ubiquitin conjugates
is obtained by performing Western blot analysis with
antibodies against ubiquitin, as described in Section
2.2.2.
2.1.3. Preparation of protein lysates by a boiling method
An alternate method to prepare lysates from FlagH2B-containing cells uses a boiling method first described by Horvath and Riezman [60]. This method has
the advantage of being rapid and using smaller volumes
of cells, although in our hands, we find that the TCA
method is generally more reproducible. A comparison of
results obtained from the two methods of lysate preparation is shown in Fig. 1.
1. Ten milliliters of cells are grown as described in Section 2.1.2 to OD600 ¼ 0.8 and collected by centrifugation.
2. The cell pellets are washed one time with distilled
water and collected again by centrifugation.
3. The cell pellets are resuspended in 160 ll of 1 Laemmli sample buffer and heated at 95 °C for 5 min.
Fig. 1. Detection of ubiquitylated Flag-H2B in yeast lysates. Lysates
were prepared from yeast cells carrying Flag-H2B or Flag-H2BK123R by a boiling method (left) or after treatment with 20% TCA
(right). Proteins were separated on a 15% acrylamide–SDS polyacrylamide gel, transferred to an Immobilon membrane, and probed with
antibody against Flag. In this and subsequent figures, blots have been
overexposed to reveal the range of background bands commonly observed.
Fig. 2. Immunoprecipitation of ubiquitylated Flag-H2B from yeast
lysates. TCA lysates were prepared from yeast cells transformed with
Flag-H2B or Flag-H2B-K123R and HA-ubiquitin, and Flag-H2B
species were immunoprecipitated by incubation with M2 agarose
beads. The immunoprecipitates were eluted by boiling, and aliquots of
each eluant were run on two 15% acrylamide–SDS polyacrylamide
gels. After transfer to Immobilon membranes, blots were probed with
antibody against Flag (left) or HA (right).
4. Following centrifugation at 14,000 rpm for 5 min,
10 ll of the lysate is loaded onto a 15% SDS–polyacrylamide gel and analyzed by Western blot analysis (Fig. 1) as described in Section 2.1.2.
2.1.4. Detection of ubiquitylated H2B in yeast chromatin
The majority of modified histones are present as
chromatin constituents. The distribution and abundance
C.-F. Kao, M.A. Osley / Methods 31 (2003) 59–66
of these modified species in chromatin have been studied
through the use of chromatin immunoprecipitation
(ChIP) assays with antibodies against specifically modified forms of the core histones, e.g., histones acetylated
at different lysine residues [61,62]. ChIP assays can also
be used to examine the chromatin association of ubiquitylated H2B. Moreover, this assay can be modified to
examine the co-association of ubiquitylated H2B with
other histone modifications, providing a powerful approach to study regulatory interactions in chromatin at
a global level [41]. The ChIP assays described below are
based on a protocol described by Kuo and Allis, with
additional modifications provided by Sun and Allis
[41,61].
1. Fifty milliliters of cells containing Flag-HTB1 or
Flag-htb1-K123R are grown to an OD600 ¼ 0.8 (40
OD600 units) in SD-tryptophan medium at 30 °C.
2. The culture is fixed by addition of 1.35 ml of 37%
formaldehyde for 20 min at room temperature, with
occasional shaking. Glycine is added to 125 mM to
quench the reaction, and incubation is continued
for an additional 5 min.
3. The fixed cells are collected by centrifugation at
5000 rpm for 5 min at 4 °C (Sorvall RC5-B), and
the cell pellet is washed twice with 25 ml of ice-cold
TBS (20 mM Tris, pH 7.4, 150 mM NaCl). The
washed pellet is quick frozen in an ethanol–dry ice
bath and stored at )80 °C.
4. The pellet is thawed on ice and resuspended in
500 ll of ice-cold lysis buffer (50 mM Hepes–KOH,
pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton
X-100, 0.1% sodium deoxycholate) that contains a
1 protease inhibitor cocktail (Sigma) plus 1 mM
PMSF (phenylmethanesulfonyl fluoride). The cell
suspension is then transferred to a 1.5-ml microfuge
tube.
5. One-half gram of acid-washed glass beads (Section
2.1.2) is added to the resuspended cells, which are
broken by vortexing at the highest setting for 30 min
at 4 °C using a TurboMix adapter (Fisher).
6. The broken cell suspension is collected by puncturing the bottom of the microfuge tube with a red-hot
No. 20 needle, followed by a quick centrifugation of
the contents, minus glass beads, into a clean 1.5-ml
microfuge tube.
7. Chromatin is sheared by sonication to an average
size of 500 bp. For example, a Branson Sonifier
250 is set at 30–40% output with a duty cycle of
90%, and sonication is performed 6 10 s on ice,
chilling the lysate in a dry ice-chilled ice bath for
30 s in between each cycle.
8. Cell debris is removed by centrifugation at
14,000 rpm for 60 min at 4 °C, and the lysate or
whole cell extract (WCE) is transferred to a clean
1.5-ml microfuge tube. This extract is used for immunoprecipitation.
63
9. The WCE is adjusted to a volume of 500 ll with icecold lysis buffer containing protease inhibitors, and
60 ll of M2 agarose beads (Sigma) is added. Incubation is continued at 4 °C for 1–2 h or overnight.
10. The agarose beads are collected by centrifugation at
14,000 rpm for 15 s at 4 °C, and the supernatant (unbound) fraction is retained to assay for the efficiency
of binding.
11. The beads are washed sequentially with 1.5 ml of the
following buffers for 5 min each at room temperature
using a rotating platform: lysis buffer; wash buffer 1
(lysis buffer containing 500 mM NaCl); wash buffer 2
(10 mM Tris–HCl, pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 1 mM EDTA); and
1 TE, pH, 8.0.
12. At this point, 100–200 ll of 1 Laemmli sample buffer is added to the beads, the Flag-H2B immunoprecipitates are released by boiling for 10 min, and the
eluted proteins are examined by western blot analysis (Fig. 3). However, to perform a second round of
ChIP with antibodies against other histone modifications, the Flag-H2B species are eluted with Flag peptide. The M2 agarose beads are resuspended in
400 ll of lysis buffer containing protease inhibitors,
and the Flag-H2B species are eluted by incubation
with 20 ll of a 4 mg/ml solution of 1 Flag peptide
(Sigma) overnight at 4 °C.
13. The eluant is now ready for immunoprecipitation
with antibodies against other modified histones
and subsequent Western blot analysis with anti Flag
antibodies. For example, using this technique, Sun
Fig. 3. Detection of ubiquitylated Flag-H2B in yeast chromatin. Yeast
cells containing Flag-H2B or Flag-H2B-K123R were fixed with
formaldehyde and chromatin was solubilized by sonication. Chromatin was incubated with M2 agarose beads, Flag-H2B species were
eluted by boiling, and Western blot analysis was performed with antibody against the Flag epitope. The asterisks represent core histones
H3, H2A, and H4 (in order of descending size) covalently linked to
Flag-H2B.
64
C.-F. Kao, M.A. Osley / Methods 31 (2003) 59–66
and Allis showed that chromatin containing histone
H3 methylated on lysine 4 is associated with chromatin that contains ubiquitylated Flag-H2B [41]. A
variety of antibodies against modified histones are
commercially available from Upstate Biotechnology,
which provides directions for immunoprecipitation
and immunoblotting.
2.2. Detection of ubiquitylated histones in vertebrate cell
lines
The first ubiquitylated protein identified in higher
eukaryotes was uH2A or A24 [9]. Early methods to
detect ubiquitylated histones relied on a combination of
one- and two-dimensional gel electrophoresis analysis,
coupled with peptide and amino acid analysis of
histones that showed modified mobility in these gel
systems [16,17,63–65]. Antibodies that specifically recognize uH2A have been described and recently have
become commercially available from Upstate Biotechnology [52]. However, to detect ubiquitylated forms of
the other histones, antibodies against ubiquitin can be
used to identify ubiquitin–protein conjugates by immunoblotting. Below, we describe a relatively simple
immunological approach to detect the presence of ubiquitylated histones in a vertebrate cell line, an approach
that is conceptually similar to the method described for
yeast. This approach employs two sets of antibodies;
those that recognize ubiquitin and those that recognize
individual core histones—and in principle the method
could be used to examine the levels and regulation of
ubiquitylated histones in a variety of higher eukaryotic
cells.
2.2.1. Acid extraction of histones from cultured HeLa
cells
In this method (modified from a published protocol
of Upstate Biotechnology), bulk histones are first extracted from cultured HeLa cells by acid extraction.
Although a number of basic nuclear proteins are released by this method, histones are enriched. The extracted proteins are then analyzed by one-dimensional
SDS–PAGE, and antibodies against both free ubiquitin
and the core histones are used to identify which histones
species are modified by ubiquitylation.
1. Hela cells cultured in DMEM (DulbeccoÕs modified
EagleÕs medium) supplemented with 10% FBS (fetal
bovine serum) are grown to 70% confluence. Typically, 10 lg of histones can be obtained from
2.5 106 cells.
2. Sodium butyrate is added to a final concentration of
5 mM to inhibit histone deacetylases, and growth is
continued for another 24 h. This step, which helps
to preserve acetylated forms of histones, is included
in case there is any ‘‘cross-talk’’ between histone
acetylation and ubiquitylation.
3. The cells are scraped from the plate and collected by
centrifugation at 1500 rpm for 2–3 min in an Heraeus
Megafuge. The cell pellet is suspended in 10–15 vol
of PBS (138 mM NaCl, 2.7 mM KCl, pH 7.4) and recentrifuged at 1500 rpm for 2 min.
4. The cell pellet is resuspended in 5–10 vol of lysis buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2 , 10 mM
KCl, 0.5 mM DTT, 1.5 mM PMSF, with PMSF
and DTT added just prior to use) in a polypropylene
tube. Ubiquitin aldehyde (Sigma) is added to a final
concentration of 5 lM to inhibit ubiquitin C-terminal hydroxylases.
5. Hydrochloric acid is added to a final concentration
of 0.2 N, and the cell suspension is incubated on
ice for 30 min.
6. The suspension is centrifuged at 11,000 rpm for
10 min at 4 °C, and the supernatant fraction, which
contains the acid soluble proteins, is retained.
7. The supernatant fraction is dialyzed twice against
200 ml of 0.1 N acetic acid for 1–2 h each, and then
dialyzed three times against 200 ml of distilled water
for 1 h, 3 h, and overnight, respectively. At this
point, the protein concentration can be determined
[22,66], and the proteins can be lyophilized or stored
at )80 °C.
2.2.2. Immunoblot analysis of acid extracted histones
1. Ten micrograms of acid extracted proteins are fractionated by 15% SDS– PAGE and transferred to a
nitrocellulose membrane (Amersham) using Tris–
glycine buffer containing 20% methanol as described
in Section 2.1.2. A duplicate gel can be stained with
Coomassie blue to identify the positions of the core
histones.
2. To ensure complete denaturation of the bound ubiquitylated proteins, the membranes are submerged in
deionized water and either autoclaved or boiled for
20 min.
3. The membranes are blocked in TBS–0.05% Tween
containing 5% milk overnight at 4 °C. At this
point, multiple strips can be incubated with antibodies against ubiquitin and antibodies against
each of the four core histones to identify which histones are modified by ubiquitin. Anti-ubiquitin
antibodies are commercially available from Sigma
and Santa Cruz. Anti-core histone antibodies with
broad species reactivity are available from Upstate
Biotechnology, which has recently added a monoclonal antibody against ubiquitylated H2A to its
catalog.
4. In a typical incubation, anti-ubiquitin antibody (rabbit polyclonal, Sigma) is diluted 1:500 into 15 ml of
TBS–Tween containing 2% milk and incubated with
the membrane for 90 min at room temperature. The
membrane is washed four times in 15 ml of TBS–
Tween for 15 min each, and then incubated with a
C.-F. Kao, M.A. Osley / Methods 31 (2003) 59–66
1:5000 dilution of goat anti-rabbit Ig (Amersham)
for 1 h in 15 ml of TBS–Tween.
5. After four 15-min washes in 15 ml of TBS–Tween,
the membrane is developed by enhanced chemiluminescence, using a kit from Bio-Rad or Perkin Elmer–
NEN according to the manufacturerÕs directions. By
comparing the mobility of bands that are recognized
by both anti-ubiquitin and anti-histone antibodies,
the identity of the ubiquitylated histones can be
established.
3. Concluding remarks
Much progress has been made in understanding the
regulation, distribution, and cellular roles of acetylated
histones because of the existence of antibodies against
histones modified on specific lysine residues. In the absence of widely available antibodies against ubiquitylated histones, other immunological approaches
outlined in this article can be effective in detecting these
forms of modified histones. Theoretically, these approaches should be sensitive enough to detect ubiquitylated histones when they are present at 1% the levels
of unmodified histones. The assays outlined have a
number of applications, particularly with respect to
studying histone ubiquitylation in a genetically tractable
organism such as budding yeast. First, they can be used
to provide an initial identification of the lysine residues
that are ubiquitylated in vivo. For example, mutation of
lysine 123 to arginine in a Flag-tagged version of yeast
H2B was shown to prevent the shift in mobility that
normally accompanies ubiquitylation of this histone in
cells [13] (Fig. 1). Second, the cellular machinery that
regulates ubiquitin conjugation to histones can be
identified. Rad6 was a leading candidate for a histonespecific ubiquitin-conjugating enzyme [44], and mutation of yeast RAD6 was shown to abolish ubiquitin
conjugation to Flag-H2B in vivo [13]. Moreover, by use
of a similar genetic/immunological approach, three yeast
proteins (Rad5, Rad18, and Ubr1) were eliminated as
histone-specific E3s, while Bre1 was identified as a
Rad6- interacting protein that regulates H2B ubiquitylation [41,53,54]. In principle, the assay system could be
used to identify other components involved in histone
ubiquitylation, such as deubiquitylating enzymes that
target histones [25]. A third use for these assays, applicable to both yeast and vertebrate cells, is to provide a
convenient measure of the global levels of ubiquitylated
histones under various conditions of cell growth and
differentiation. For example, ubiquitylated Flag-H2B
was found to be present at mitosis during the yeast cell
cycle (S. Gladden and M.A. Osley, unpublished data),
and Flag-H2B was shown to become ubiquitylated when
yeast cells enter meiosis [13]. Finally, the assays can be
adapted to study regulatory interactions between dif-
65
ferent types of histone modifications. Flag-H2B was
shown to be ubiquitylated in yeast in the absence of
Set1, a methyltransferase that modifies H3 on lysine 4,
but in the absence of H2B ubiquitylation (e.g., in an
H2B-K123R or rad6D mutant), H3 Lys 4 could not be
methylated [41]. By use of a similar approach, this unidirectional, trans-tail regulation by ubiquitylated H2B
was shown to apply to the methylation of selected lysine
residues in H3 [40,42].
Despite the wide range of biological questions that
can be asked with epitope-tagged histones and/or
ubiquitin, the assays outlined in this article are applicable only for studies that examine the global levels of
ubiquitylated histones. To determine the precise cellular
distribution of these modified histones in chromatin,
such as at particular promoters during gene activation
or repression, antibodies that specifically recognize ubiquitylated histones will have to be employed. When
such antibodies become available, and if they function in
chromatin immunoprecipitation assays, it will be very
exciting to learn if histone ubiquitylation, like histone
acetylation, is regulated on a local as well as a global
level.
Acknowledgments
Judith Recht and Kenneth Robzyk are thanked for
their seminal contributions to the detection and identification of uH2B in yeast. Zu-Wen Sun is gratefully
acknowledged for providing the protocol for the immunoprecipitation and elution of Flag-H2B from yeast
chromatin, Pamela Meluh is thanked for her modifications of yeast lysate preparations, and Rosa Bermudez is
thanked for technical information. The work in this
article from the authorsÕ laboratory was supported by
Grant GM40118 from the NIH.
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