Recombination-induced tag exchange to track old and new proteins.
ABSTRACT The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods.
- SourceAvailable from: link.springer.com[show abstract] [hide abstract]
ABSTRACT: It has long been known that epigenetic changes are inheritable. However, except for DNA methylation, little is known about the molecular mechanisms of epigenetic inheritance. Many types of stem cells undergo asymmetric cell divisions to generate self-renewed stem cells and daughter cells committed for differentiation. Still, whether and how stem cells retain their epigenetic memory remain questions to be elucidated. During the asymmetric division of Drosophila male germline stem cell (GSC), our recent studies revealed that the preexisting histone 3 (H3) are selectively segregated to the GSC, whereas newly synthesized H3 deposited during DNA replication are enriched in the differentiating daughter cell. We propose a two-step model to explain this asymmetric histone distribution. First, prior to mitosis, preexisting histones and newly synthesized histones are differentially distributed at two sets of sister chromatids. Next, during mitosis, the set of sister chromatids that mainly consist of preexisting histones are segregated to GSCs, while the other set of sister chromatids enriched with newly synthesized histones are partitioned to the daughter cell committed for differentiation. In this review, we apply current knowledge about epigenetic inheritance and asymmetric cell division to inform our discussion of potential molecular mechanisms and the cellular basis underlying this asymmetric histone distribution pattern. We will also discuss whether this phenomenon contributes to the maintenance of stem cell identity and resetting chromatin structure in the other daughter cell for differentiation.Chromosome Research 05/2013; 21(3):255-69. · 2.85 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Following mitosis, daughter cells must inherit a functional set of essential proteins and organelles. We applied a genetic tool to simultaneously monitor the kinetics and distribution of old and new proteins marking all intracellular compartments in budding yeasts. Most organelles followed a general pattern whereby preexisting proteins are symmetrically partitioned followed by template-based incorporation of new proteins. Peroxisomes belong to this group, supporting a model of biogenesis by growth and division from preexisting peroxisomes. We detected two exceptions: the nuclear pore complex (NPC) and the spindle pole body (SPB). Old NPCs are stably inherited during successive generations but remained separated from new NPCs, which are incorporated de novo in mother and daughter cells. Only the SPB displayed asymmetrical distribution, with old components primarily inherited by daughter cells and new proteins equally incorporated in both cells. Our analysis resolves conflicting models (peroxisomes, NPC) and reveals unique patterns (NPC, SPB) of organelle inheritance.Proceedings of the National Academy of Sciences 12/2012; · 9.74 Impact Factor
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ABSTRACT: Proteins are not static entities. They are highly mobile and their steady state levels are achieved by a balance between ongoing synthesis and degradation. The dynamic properties of a protein can have important consequences for its function. For example, when a protein is degraded and replaced by a newly synthesized one, post-translational modifications are lost and need to be reincorporated in the new molecules. Protein stability and mobility are also relevant for duplication of macromolecular structures or organelles, which involves coordination of protein inheritance with the synthesis and assembly of newly synthesized proteins. To measure protein dynamics we recently developed a genetic pulse-chase assay called Recombination-Induced Tag Exchange (RITE). RITE has been successfully used in Saccharomyces cerevisiae to measure turnover and inheritance of histone proteins, to study changes in post-translational modifications on aging proteins, and to visualize the spatiotemporal inheritance of protein complexes and organelles in dividing cells. Here we describe a series of successful RITE cassettes that are designed for biochemical analyses, genomics studies, as well as single cell fluorescence applications. Importantly, the genetic nature and the stability of the tag-switch offer the unique possibility to combine RITE with high-throughput screening for protein dynamics mutants and mechanisms. The RITE cassettes are widely applicable, modular by design, and can therefore be easily adapted for use in other cell types or organisms.G3-Genes Genomes Genetics 05/2013; · 1.79 Impact Factor
Recombination-induced tag exchange to track old and
Kitty F. Verzijlbergena, Victoria Menendez-Benitob, Tibor van Welsema, Sjoerd J. van Deventerb, Derek L. Lindstromc,
Huib Ovaab, Jacques Neefjesb, Daniel E. Gottschlingc, and Fred van Leeuwena,1
aDivision of Gene Regulation andbDivision of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; andcDivision of Basic Sciences,
Fred Hutchinson Cancer Research Center, Seattle, WA 98109
Edited by Michael Grunstein, David Geffen School of Medicine at UCLA, Los Angeles, CA, and approved November 17, 2009 (received for review September
However, analyzing dynamics of proteinsand protein complexesin
vivo has been difficult. Here we describe recombination-induced
tag exchange (RITE), a genetic method that induces a permanent
activation of Cre recombinase. The time-controlled tag switch pro-
vides a unique ability to detect and separate old and new proteins
in time and space, which opens up opportunities to investigate the
dynamic behavior of proteins. We validated the technology by de-
termining exchange of endogenous histones in chromatin by bio-
chemical methods and by visualizing and quantifying replacement
of old by new proteasomes in single cells by microscopy. RITE is
widely applicable and allows probing spatiotemporal changes in
protein properties by multiple methods.
translational processing, modification, and demodification. In
addition, most proteins are very mobile and undergo interactions
with multiple other protein partners (1–4). However, little is
known about the dynamics of proteins within macromolecular
complexes in vivo (2, 4). Studying time-dependent changes in
physical properties of proteins or protein turnover requires
methods to distinguish resident (old) proteins from new proteins.
Current methods that do so are usually based on fluorescent re-
porters or differential chemical labeling. For example, fluo-
rescence recovery after photo bleaching relies on exchange of the
old bleached protein by nonbleached proteins (1, 3, 4). Alter-
native methods involve time-dependent changes in fluorescence,
nonspecific pulse-chase labeling of proteins with labeled amino
acids, or labeling with chemical dyes that specifically bind to short
tags (5–7). Although suitable for detection of proteins by micro-
scopy or mass spectrometry, a limitation of these methods is that
they do not provide a handle for biochemical analysis of old and
new proteins and their complexes. To solve this problem and to
eliminate the requirement for chemical labels or UV light
we developed recombination-induced tag exchange (RITE), a
method in which a genetic epitope tag is switched by transient
induction of a site-specific recombinase. As a consequence, old
and newly synthesized proteins are differentially tagged, which
illustrated here. In contrast to inducible expression strategies (8–
12), differential tagging by a time-controlled site-specific protease
(13), or the labeling methods described above, RITE allows par-
allel detection and purification of old and new proteins under
physiological conditions and over long periods of time.
We used RITE to probe the stability of chromatin. Photo-
bleaching experiments using histones tagged with fluorescent
recent work suggests that chromatin is more dynamic than pre-
can be incorporated into chromatin of nondividing yeast cells and
roteins are dynamic molecules. Their abundance is controlled
by synthesis and degradation and they can be subject to post-
gene activation of certain promoters is accompanied by transient
loss of histones (8–12, 16). In metazoans, the histone H3 variant
transcription-coupled process (17–19). We took advantage of
RITE to determine whether endogenously expressed canonical
histones undergo replication-independent exchange. RITE can
also be used to visualize proteins by microscopy. To demonstrate
this we applied RITE to the proteasome, a highly conserved and
essential macromolecular complex critical for degradation of
proteins by proteolysis (20). Using fluorescent RITE we could
and cytoplasm of dividing cells.
RITE Outline. RITE can be applied by integration of a RITE cas-
settedownstream ofany gene ofinterest, resulting in aC-terminal
tag situated between two LoxP sites with an orphan tag down-
stream. Upon a transient time-controlled activation of the site-
specific Cre recombinase, recombination between the tandem
LoxP sitesresults in exchange ofthe “old”tag by an orphan “new”
tag in the coding sequence leading to an epitope-tag switch (Fig.
1). After switching, all newly synthesized mRNAs will encode for
proteins containing thenew epitope tag.The LoxPrecombination
sites are part of the coding sequence, which eliminates the need
for introns and allows the tag cassette to be introduced directly at
consequence, the differentially tagged proteins are encoded by a
single gene and under control of the endogenous promoter. Re-
combination can be induced using a constitutively expressed Cre
recombinase fused to the human estrogen binding domain
(EBD). This fusion protein is sequestered by heat-shock proteins
and inactive (21). The nuclear activity of Cre-EBD can be rapidly
activated by the addition of β-estradiol, which releases the fusion
protein from heat-shock proteins (21). A major advantage of
RITE is that the genetic switch is permanent. Therefore, after the
switch both old and new proteins can be followed in the original
cells and their descendants under any condition of interest. We
applied this strategy in haploid yeast cells and integrated RITE
cassettes by homologous recombination at endogenous gene loci.
Application of RITE to Histone H3. FirstRITEwasappliedtohistone
Author contributions: J.N., D.E.G., and F.v.L. designed research; K.F.V., V.M.-B., T.v.W., and
S.J.v.D. performed research; D.L.L. and H.O. contributed new reagents/analytic tools; K.F.V.,
V.M.-B., S.J.v.D., J.N., and F.v.L. analyzed data; and K.F.V., V.M.-B., and F.v.L. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed at: Division of Gene Regulation, Nether-
lands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail:
This article contains supporting information online at www.pnas.org/cgi/content/full/
| January 5, 2010
| vol. 107
| no. 1 www.pnas.org/cgi/doi/10.1073/pnas.0911164107
the two histone H3 genes was tagged with a RITE cassette con-
taining two small epitope tags, HA and T7 (H3-HA→T7) (Fig.
2A). The second histone H3 gene was deleted. As a consequence,
in this strain all histone H3 proteins were tagged (Fig. 2A). Yeast
cells expressing the tagged histones are viable (Fig. 2B). Because
histone H3 is essential, this demonstrates that the tagged H3
which has no detectable effect on growth or transcription (22),
most of the cells had undergone recombination within 2 h (Fig.
2C). To confirm that the genetic switch at the DNA level yields
differentially tagged proteins, switched starved cells (see below)
were released in fresh media and harvested at several time points
after reentry into the cell cycle. Immunoblot analysis demon-
strated replacement of old histone H3-HA protein by new H3-T7
in dividing cells (Fig. 2D). The replacement of one tagged protein
by the other is in contrast to previously used “inducible-
expression” strategies, which involve ectopic expression of a tag-
ged (new) version of a protein by an inducible promoter in the
presence of an endogenous copy. Because of ongoing synthesis of
the endogenous gene copy, endogenous histones represent old as
well as new proteins. As a consequence, the induced and endog-
enous proteins quickly reach a new steady state. Tagging a single
endogenous gene with a RITE cassette eliminates this problem
and allows simultaneous tracking of old and new proteins over
many cell divisions.
Cells.Quantification of the immunoblot shown in Fig. 2D showed
that replacement of old H3-HA by new H3-T7 occurred at a rate
faster than expected when only dilution due to replication is
taken into account, suggesting histone turnover by replication-
independent mechanisms (Fig. 2E). The fact that RITE in-
troduces a permanent genetic switch after a transient signal al-
lowed direct comparison of histone exchange in different cell
cycle stages. To minimize new histone mRNA and protein ex-
pression during the recombination process the tag switch was
performed in nutrient-starved cells, here referred to as G0 (Fig.
3A and Fig. S1). Switched H3-HA→T7 cells were released into
fresh medium containing nocodazole to arrest the cells after
passage through one S-phase in G2/M (Fig. 3A and Fig. S2).
During S-phase, like the DNA, the amount of histones gets du-
plicated and incorporated into the chromatin. As expected, cells
settes contain two epitope tags (old and new), the first of which is in be-
tween two LoxP sites. Integration of a RITE cassette downstream of an ORF
(ORF) results in a protein tagged with an old tag (blue). The old tag is pre-
ceded by an invariant flexible spacer (S) and a short peptide encoded by the
LoxP sequence (LoxP) and is followed by a transcriptional terminator (stop)
and a selectable marker (select). Upon induction of Cre recombinase, site-
specific recombination between the tandem LoxP sites in the genome results
in loss of the old tag and fusion of the ORF to the new tag. After the switch,
newly synthesized proteins will contain the new tag (yellow), whereas ex-
isting proteins will contain the old tag. Old and new proteins are expressed
from the same gene by the native promoter.
Outline of recombination-induced tag exchange (RITE). RITE cas-
genes encoding histone H3 in yeast (HHT2) was tagged with a RITE cassette
(H3-RITE) containing short epitope tags: HA (old) and T7 (new). The other
gene encoding histone H3 (HHT1) was deleted. A Hygromycin resistance
gene (Hygro) was used to select against illegitimate recombinants. The tag
switch was under control of a constitutively expressed hormone-dependent
Cre recombinase (Cre-EBD78). (B) Growth of wild-type and H3 RITE-tagged
[before (HA) and after (T7) the switch] yeast cells spotted in a 10-fold dilu-
tion series. (C) The efficiency of recombination in the cell population was
determined by Southern blot analysis of genomic DNA digested with HindIII
(H) before (Pre) and after (Post) addition of the hormone β-estradiol. An
invariant fragment was used as a control (Ctrl). (D) Detection of old (HA) and
new (T7) histone H3 by quantitative immunoblot analysis of whole cell ly-
sates of equal numbers of starved switched cells released into fresh media.
The number of population doublings was calculated by staining the cells
with N-hydroxysuccinimide-tetra-ethylrhodamine (NHS-TER) (SI Materials
and Methods). (E) The percentage of old H3-HA plotted against the number
of population doublings. The measured HA/T7 ratios of the blot in D were
converted into H3-HA percentages by using standard curves of samples with
known percentages of H3-HA and H3-T7 (SI Materials and Methods).
Application of RITE to endogenous histone H3. (A) One of the two
strains were grown tosaturation(herereferred toas G0)incompletemedium
and recombination was induced overnight (switch) by addition of hormone
(Fig.S1).Cellswere releasedinfresh mediaandarrestedinG1(α-factor)orG2/
M (nocodazole). Samples were taken at the estimated start of the arrest (2 h
G1 and 3 h G2/M) and 3 h later. (B) Quantitative immunoblot analysis of old
and new histone H3 in whole-cell lysates using antibodies against HA (old), T7
(new) or an antibody raised against the spacer-LoxP sequence (LoxP) recog-
nizing old and new proteins simultaneously. (C) Relative H3-T7/H3-HA ratios
(New/Old) were calculated on the basis of the ratio of the top band (H3-HA)
and the bottom band (H3-T7) of the LoxP blot (absolute values) and the ratio
of HA and T7 signals (arbitrary units).
Global histone exchange determined by immunodetection. (A) Yeast
Verzijlbergen et al.PNAS
| January 5, 2010
| vol. 107
| no. 1
at the estimated start of the G2/M cell cycle block (t = 3 h)
showed an approximately equal abundance of old H3-HA and
new H3-T7 (Fig. 3 B and C). To investigate replication-in-
dependent histone exchange, the switched H3-HA→T7 cells were
released into fresh media containing α-factor to arrest the cells in
G1, to prevent passage through S-phase (Fig. 3A). New H3-T7
was detected at the start of the cell block (t = 2 h) and increased
further during the next 3 h (t = 5 h). Moreover, the abundance of
new histone H3-T7 after 5 h in G1 was similar to that of cells
arrested in G2/M, which had undergone one round of genome
duplication and therefore contain at least 50% new H3-T7 and
50% old H3-HA (Fig. 3 B and C). Thus, yeast cells that had been
arrested in G1 for the duration of around three cell doubling
times had replaced approximately half of the old H3-HA protein
by new H3-T7 in the absence of DNA replication.
Affinity Purification of Old and New Histones in Chromatin. Because
soluble histones represent a minor fraction of the total histone
pool (23), these results suggested that the G1-arrested cells had
incorporated new histone H3-T7 into chromatin. To address this
question we took advantage of the possibility of using the epitope
tags for affinity purification of chromatin fragments containing
old and new histones. Following chromatin immunoprecipitation
(ChIP) the ratio of new H3-T7 over old H3-HA was determined
by real-time quantitative PCR (qPCR) for promoter regions of a
set of genes with different transcriptional properties and for an
intergenic region (Fig. 4A). Histone exchange in chromatin was
already detectable in switched G0 cells before release. After
supplementation of fresh medium containing α-factor, exchange
increased in the transition to the G1 arrest and increased further
during the arrest (2 and 5 h G1). Strikingly, 5 h after release into
the G1 block, the replacement of old H3-HA by new H3-T7 was
quantitatively similar at different loci to that of cells that had just
duplicated their genome and histone content (3 h G2/M). This
confirms that cells arrested in G1 had undergone rapid replication-
independent exchange of chromatin-bound histones (Fig. 4A).
However, histone exchange was not restricted to the G1 phase.
Cells arrested in G2/M (from 3 h until 6 h) and even cells arrested
by nutrient depletion (G0 pre until G0 post) accumulated new
H3-T7 duringthe arrest, albeit slower(Figs. 3C and4A).Identical
results were obtained with a strain in which the old and new tags
were swapped (H3-T7→HA; Fig. S3), showing that the charac-
teristics of new histone deposition were not determined by the
specific epitope tags. We conclude that replication-independent
histone exchange is a common feature of arrested cells but the
rate of exchange can vary between cell cycle phases.
RITE allowed a direct and quantitative comparison between
G1 and G2/M cells, which demonstrated that cells arrested in
G1 replaced half of the old histones by new histones within 5 h
by replication-independent mechanisms. Analysis of mRNA ex-
pressionlevels duringthedifferent phasesofthecell cycleshowed
that the rate of histone exchange was coupled to the level of
(Fig. 4B and Fig. S4). Analysis of the inducible GAL1 promoter
showed that induction of transcription caused an increase in his-
tone exchange (Fig. S4), suggesting that transcription leads to
histone exchange. In addition, transcription-coupled histone ex-
found at promoters (Fig. S5). Transcription-coupled histone ex-
change might be a specific property of arrested cells that cannot
replace histones by replication-dependent mechanisms. To in-
vestigate this possibility, histone exchange in chromatin was de-
termined in log-phase cells that had been grown for many
generations without a growth arrest (Fig. 4C). In these cycling
highly transcribed genes (Fig. 4C), suggesting that transcription-
histone deposition. In addition, monitoring of old and new histo-
nes during successive cell divisions showed that transcription-
coupled histone deposition was maintained during at least three
cell divisions (Fig. S6). Thus, biochemical purification of old and
new histones revealed that chromatin is a very dynamic macro-
molecular complex in dividing as well as nondividing cells and
that transcription is a key determinant of chromatin instability.
Fluorescent RITE to Monitor Proteasome Replacement in Time and
Space. The methods discussed above probe protein dynamics in
pools of cells. To visualize the behavior of old and new proteins in
single cells, a fluorescent RITE cassette was constructed that
switches from a green fluorescent protein (GFP) tag to a mono-
meric red fluorescent protein (mRFP) tag (Fig. 5A). To illustrate
the use of fluorescent RITE, a constituent protein of another
macromolecular complex, the proteasome, was tagged. Specifi-
cally, we constructed a yeast strain where the only endogenous
PRE3 gene, encoding a catalytic β-subunit of the proteasome, was
quantified by affinity purification. (A) Analysis of chromatin-bound histones
by ChIP of HA (old) and T7 (new) histone H3 quantified by real-time quan-
titative PCR (qPCR). Histone exchange (ratio of new/old) was determined for
promoters of the indicated genes and an intergenic region on chromosome
V (NoORF). The genes are ranked by estimated transcription frequency in log
phase, from low (open bars) to high (solid bars) frequency. The result shown
is the average of two individual experiments (± SEM). The Inset is a zoom-in
of the G0 time points. (B) Relative mRNA expression levels were determined
by reverse-transcriptase qPCR (RT-qPCR). An S-phase sample of the same H3-
RITE strain was used as a reference sample. A wild-type strain without a RITE
tag showed very similar expression profiles (Fig. S4). (C) Histone turnover
in H3-T7→HA cells without any arrest was determined by induction of Cre-
recombinase in log-phase cells (OD660= 0.25). The percentage of cells that
had undergone recombination (Rec) is indicated for each time point (de-
termined by a colony-plating assay). Histone replacement at promoters was
determined by ChIP (HA/T7).
Replication-independent transcription-coupled histone turnover
| www.pnas.org/cgi/doi/10.1073/pnas.0911164107 Verzijlbergen et al.
tagged with the GFP→mRFP RITE cassette. This strain has a
normal growth rate, indicating that the RITE-tagged subunit is
functional, because deletion or mutation of PRE3 is lethal. We
note that yeast cells expressing H3-GFP→mRFP were inviable,
indicating that not every protein can be safely tagged with the
larger GFP→mRFP RITE cassette. To visualize the replacement
of old by new proteasomes by microscopy, recombination was
induced in G0 (Fig. 5B), during which very little proteasome
synthesis occurs. Because the proteasome is a stable complex,
many old proteasomes (Pre3-GFP) remain that are slowly re-
placed by new proteasomes (Pre3-mRFP) (Fig. 5C). When the
swiftly replaced by new proteasomes due to dilution during cell
present in both the nucleus and the cytosol (24). Quantification of
GFP and mRFP signals showed that in the switched yeast cells,
the appearance of new proteasome and loss of old proteasome
followed similar kinetics in the two compartments (Fig. 5D). Thus
fluorescent RITE enables visualization of replacement of old by
new proteins in living cells in time and space during cell cycle
arrests and during successive cell divisions.
Here we show that RITE is a versatile method to study different
parameters of protein dynamics such as protein turnover and
other methods such as pulse-chase labeling, inducible expression,
methods based on differential fluorescence, or TimeStamp (3, 5,
6, 13, 25), RITE provides the unique possibility to simultaneously
monitoroldandnewproteinsandtodo soby multipletechniques.
RITE has important additional advantages over existing tech-
nologies. It does not require addition of UV light, chemicals, or
labels, circumventing the need for expensive ultrasensitive mass
spectrometry technologies. Furthermore, because no heterolo-
andnew proteins, taggedgenes areregulated by their endogenous
promoter and the switch can occur without perturbation under
any condition of interest. Protein replacement of the stable pro-
teasomes and histones could be assessed over long timeperiods in
dividing and nondividing cells, indicating that RITE is suitable to
study the dynamics of long-lived proteins, which are typically
∼2 h until the majority of the cells has switched, switched cells can
already be detected as early as 15 min after activation of Cre.
RITE may be less suitable for studies of very short-lived proteins.
The differential tagging of histone H3 showed that endoge-
nously expressed canonical histones undergo turnover within
chromatin in a transcription-dependent manner. Our results are
in agreement with previous histone H3 turnover studies using
time-controlled induced expression of a tagged ectopic histone
copy in yeast (8–12, 16, 26). The direct comparison with repli-
cation-dependent assembly of new histones indicates that repli-
cation-independent histone exchange occurs at a high rate. This
was unexpected when one considers the regulated expression of
histones. We note that whereas H3 mRNA indeed peaks in
by microscopy. (A) Schematic representation of fluorescent
RITE. (B) PRE3-GFP→mRFP cells were grown to saturation (G0)
and recombination was induced overnight (switch). Sub-
sequently, cells were released in fresh media (release 1) and
samples were taken at the indicated time points. Nine hours
after the first release, cells were again supplemented with
fresh media (release 2). Time points 3, 6, 9, and 24 h corre-
spond to ≈0.3, 2, 3, and 8 cell divisions, respectively. (C)
GFP→mRFP grown as indicated in B and of control strains
(PRE3-GFP and PRE3-mRFP). Hoechst was used as a nuclear
counterstaining (blue). (Scale bar, 4 μm.) (D) The GFP and
mRFP fluorescent intensities of micrographs from C were
quantified and the value shown for each time point is an
average of the mean fluorescence intensity in the nuclei, cy-
toplasm, and total surface of 400 cells (± SD). Dashed lines
indicate GFP and mRFP signals in control cells expressing GFP
or mRFP only (the bottom dashed lines indicate background
Spatiotemporal analysis of old and new proteasomes
Verzijlbergen et al. PNAS
| January 5, 2010
| vol. 107
| no. 1
S-phase when chromatin is duplicated, its expression is lower but
still substantial outside of S-phase (Fig. 4B). This supports the
idea that canonical histones are synthesized outside of S-phase
for replication-independent histone exchange. Especially in
starvedcells, H3 mRNA is relatively abundant (Fig.4B). The high
rate of histone exchange suggests that posttranslational mod-
ifications in chromatin are continuously being erased in dividing
and nondividing cells. Thus, replication-independent histone
exchange might provide cycling and noncycling cells with a means
to replace old histones that have acquired damage or that need to
be epigenetically reset.
Using fluorescent RITE, replacement of old by new protea-
somes in time and space was determined by microscopy. The
amount of old proteasomes decreased at a very similar rate in the
cytosolic and nuclear compartments, suggesting an even segre-
gation during cell division and/or a fast reequilibration between
proteasomes in both compartments. Likewise, the appearance of
new proteasome in both compartments followed similar kinetics,
indicating that the translocation of new proteasome subunits into
the nucleus is a relatively fast phenomenon (Fig. 5D).
RITE is a widely applicable tool to dissect novel mechanisms
and functions of protein dynamics. For example, RITE-tagged
genes of interest and the Cre recombinase can be efficiently
introduced into the collection of yeast deletion strains by one
round of genetic crossing, which allows genomewide genetic
screens for identification of factors involved in protein dynamics.
RITE can also be applied to investigate whether new and aging
proteins have different properties such as age-related post-
translational modifications or whether they show differential
segregation between mother and daughter cells. Finally, although
we have validated RITE in budding yeast, with minor mod-
ifications RITE technology may be adapted for use in higher
eukaryotes. The RITE cassettes are universally applicable and
conditional versions of Cre recombinase have already been
developed for many cell systems or even whole organisms (27).
Materials and Methods
Yeast Strains and Growth Conditions. Yeast strains and growth conditions are
described in Table S1 and SI Materials and Methods. RITE cassettes contain
an invariant short peptide spacer sequence (GGSGGS) that was found to be
required for viability of strains carrying tagged histones. The spacer and
ITSYNVCYTKLS peptide encoded by the LoxP DNA sequence are present in
front of the epitope tags both before and after the switch. RITE cassettes
were PCR amplified and targeted to the 3′ end of the endogenous genes by
homologous recombination to tag the C terminus and ensure regulation by
the endogenous promoter. The hormone-dependent Cre-EBD (Cre-EBD78)
was described previously (22). A constitutively expressed copy was stably
integrated in the yeast genome. For RITE experiments, yeast cells were
grown overnight in YPD in the presence of Hygromycin B (200 μg/mL, In-
vitrogen). The cells were then diluted 1:10 into fresh YPD and incubated for
30–36 h. Recombination was induced by the addition of 1 μM β-estradiol
(E-8875, Sigma-Aldrich). Subsequently, cells were diluted 1:25 in fresh YPD
media to release the cells back into the cell cycle. Cells enter G1 arrest upon
addition of 0.5 ng/μL of α-factor and G2/M arrest upon addition of 15 μg/mL
Nocodazole (Sigma-Aldrich). Detailed protocols for ChIP, RT-PCR, immuno-
blot, Southern blot, FACS, and microscopy are described in SI Materials and
Methods and Table S2.
ACKNOWLEDGMENTS. We thank C. Logie for helpful suggestions; F. van
Diepen, A. Pfauth, and L. Oomen for technical assistance; and G. Filion for
statistical help. We thank members of the van Leeuwen lab and M. Fornerod
for suggestions and critical reading of the manuscript. F.v.L. was supported by
the European Union 6th framework program (Network of Excellence “The
Epigenome” LSHG-CT-2004-503433) and by The Netherlands Organization for
Scientific Research. D.L.L. was supported by postdoctoral fellowship PF-04-041-
01-GMC from the American Cancer Society. V.M.B. was supported by a long-
term European Molecular Biology Organization fellowship.
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| www.pnas.org/cgi/doi/10.1073/pnas.0911164107 Verzijlbergen et al.
Fred van Leeuwen