From the Institute of Microbial Technology, Sector 39A,
Chandigarh-160 036, India and the Department of
Pathology, Stanford University, Stanford, Californina 94305-5324
Received for publication, December 13, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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Mating-type silencing in
Schizosaccharomyces pombe is brought about by cooperative
interactions between cis-acting DNA sequences flanking
mat2P and mat3M and the trans-acting factors,
namely Swi6, Clr1-Clr4, Clr6, and Rik1. In addition, DNA repair gene rhp6, which plays a role in post-replication DNA repair and
ubiquitination of proteins including histones, is also involved in
silencing, albeit in a unique way; its effect on silencing and
chromatin structure of the donor loci is dependent on their switching
competence. Earlier, we hypothesized the existence of a mediator of
Rhp6 that plays a role in reestablishment of the chromatin structure
coincidentally with DNA replication associated with mating-type
switching. Here we report the identification of a 22-kDa protein as an
in vivo target and mediator of Rhp6 in mating-type
silencing. The level of this protein is greatly elevated in
sng1-1/rhp6 Mating-type silencing in two distantly related yeasts,
Saccharomyces cerevisiae and Schizosaccharomyces
pombe, occurs by analogous mechanisms with the participation of
cis-acting DNA sequences that flank the silent loci and trans-acting
factors (1, 2). Early studies in S. cerevisiae showed that
cis-acting sequences, which include an autonomous replication consensus
sequence and binding sites for ABF1 and RAP1, are important for
silencing (for review see Ref. 1). In addition, genes encoding mating
regulator/SIR1 proteins
(3-5) are also involved in silencing. Genetic and biochemical studies
have shown a role of interaction between the N terminus of histone H4
and SIR3 in silencing (6-8). In parallel studies, mutations in origin
recognition complex subunits were found to result in loss of silencing
(9-12). These results, together with the earlier work of Miller and
Nasmyth (13) and Rivier and Rine (14), suggested a coupling between
establishment of silent chromatin structure and DNA replication. In
addition, mutations in yeast homologues of chromatin assembly factor
also cause a defect in silencing (15, 16). However, recent studies have
shown that DNA replication could be decoupled from silencing in strains
where SIR1 protein could be recruited artificially (17, 18), rather than by origin recognition complex (19).
In S. pombe, mutations in swi6,
clr1-clr4, clr6, and rik1 have been
shown to abrogate silencing (20-24). Swi6p contains the conserved
chromodomain and chromo-shadow domain (20). Likewise, Clr4p contains
the SET domain (25), which is associated with methyltransferase
activity directed toward Lys-9 in histone H3 (26, 27), whereas
clr3 and clr6 encode histone deacetylases (24).
An orthologue of SIR2 has also been found in S. pombe indicating the conservation of heterochromatin components in these two
yeasts and higher eukaryotes (28, 29).
Among cis-acting sequences, the K region spanning the
mat2-mat3 interval is required for establishing a
switching/silencing-competent state (30). Strains deleted for this
region, which includes a stretch of strong homology to the centromere
repeats (31), exhibit two alternative states of switching and silencing
of a ura4+ reporter gene, which are not only
stably inherited mitotically but also segregate as "Mendelian"
epi-alleles during meiosis (30). Furthermore, the dosage of Swi6 is
critical for maintenance of these alternative epi-alleles (32). More
importantly, the requirement of cis-acting sequences with autonomous
replication sequence-like function, for silencing (33, 34), suggests an
involvement of DNA replication in silencing in S. pombe.
Very recently, DNA polymerase It was shown recently that sng1-1, a mutation in DNA repair
gene rhp6 also abrogates silencing (38). However, the effect of this mutation is unique; unlike the swi6 and
clr1-4 mutations (20-23), it causes derepression of silent
loci only if they are switching-competent (38). It was proposed that
Rhp6 plays a role during replication associated with switching in
reestablishment of silent chromatin structure at the switching donor
loci (38, 39). Likewise, a role of Rad6 in silencing at telomere and
mating-type loci was reported in S. cerevisiae (40). Rad6p
can ubiquitinate histones in vitro (41) and other proteins
and target them to degradation by proteasome according to the N-end
rule (42). Recently, in vivo RAD6-dependent
ubiquitination of histone H2B has been demonstrated in S. cerevisiae (43). However, this modification is essential for
sporulation, not for silencing (43). In mice, disruption of one of the
two copies of the RAD6 homologue (HHR6B) leads to male sterility, as
HHR6B-dependent chromatin modification and nucleoprotein
transition during spermatogenesis fail to occur (44). Thus, an in
vivo target(s) for Rhp6/Rad6 that mediates its role in silencing
remains to be discovered.
We have sought to identify the hypothetical target/mediator of Rhp6 in
silencing. Earlier, we proposed such a mediator to function transiently
during assembly of chromatin at the silent loci, following which it is
ubiquitinated and degraded (38, 39). In this study, we have identified
a 22-kDa protein as a mediator of Rhp6; this protein contains the
histone-fold motif, interacts with histone H2B in vitro, and
is subject to ubiquitination in vivo. Accordingly, the
protein has been renamed as ubiquitinated histone-like protein, Uhp1.
Both overexpression and deletion of the uhp1 gene abrogate
mating-type silencing, thus demonstrating its role as a mediator of
Rhp6 in assembly of heterochromatin and silencing.
Media and Strains--
Media were prepared according to Moreno
et al. (45). To check sporulation levels, strains were grown
at 30 °C on PMA or PMA-leu plates (in case of selection for plasmid
with LEU2 marker) for 4 days; colonies were stained with iodine for
2-3 min (45) and photographed. For quantitation of sporulation levels,
cells were examined by light microscopy, and the number of zygotic and haploid meiosis asci were counted. Strains used in this study and their
genotypes are listed in Table I.
Protein Extraction from Yeast Cells--
Cultures of appropriate
strains were grown up to log phase, harvested, and washed with Buffer A
(0.02 M HEPES, 0.1 M NaCl, 2 mM
EDTA, 0.625% glycerol, and 1 mM Purification of the 22-kDa Protein--
Because the level of the
22-kDa protein is elevated in the rhp6 V8 Protease Digestion and Microsequencing--
20 µg of the
electroeluted Uhp1 was digested with V8 protease (which specifically
cleaves peptide bonds at the C-group of acidic amino acid residue
glutamic acid in ammonium carbonate buffer, pH 7.8, and glutamic acid
and aspartic acid in sodium or potassium phosphate buffer, pH 7.8) in a
reaction mix of 200 µl containing 50 mM sodium phosphate
buffer, pH 7.8, 2 mM EDTA, and 1.2 µg of V8 protease at
37 °C for 24 h. The reaction mix was passed through a 5-kDa
cut-off filter (Amicon). Samples were then subjected to SDS-PAGE and
silver-stained. The larger aliquot retained after V8 digestion was also
subjected to SDS-PAGE in parallel lanes and electroblotted to
polyvinylidene difluoride membrane using 10 mM CAPS buffer.
Membrane was stained with 0.1% Coomassie Blue stain in 50% methanol
for 5 min. The required bands were excised, destained in 50% methanol
and 10% acetic acid (10-20 min), washed with deionized water, dried,
and subjected to microsequencing using model 470 A Protein Sequencer
(Applied Biosystems) equipped with an on-line PTH analyzer.
Construction of GST-histone and HA-Uhp1 Constructs--
Genes
encoding histones H2A, H2B, and H3 were PCR-amplified from genomic DNA
with specific primers, as shown in Table
II, and cloned at BamHI and
EcoRI sites of the vector pGEX-2T (Amersham Biosciences).
Similarly, uhp1 gene was PCR-amplified using specific primers shown in Table II and cloned at the NotI site of the
vector pREP-1N-3HA (a kind gift of M. Yanagida), in which
nmt1 promoter drives the expression of HA-tagged
uhp1 gene. The resulting vector is called pHA-Uhp1 (Table
I), in which there is an attachment of a C-terminal triple HA epitope
tag to Uhp1.
Preparation of RNA, Northern Blotting, and Reverse Transcription
(RT)-PCR--
RNA was prepared from the required strains as described
by Schmitt et al. (48). Conditions of RT-PCR for detecting
mat2Pc, mat3Mi, and pol Protein Extraction from E. coli Strains Expressing Recombinant
Plasmids--
A freshly grown single colony of Escherichia
coli strain JM109 expressing recombinant plasmid was inoculated
into 2-5 ml of LB-ampicillin medium and incubated overnight at
37 °C. This culture was reinoculated into 25-100 ml of
LB-ampicillin medium and grown at 37 °C.
Isopropyl-1-thio- Antibody Production--
For raising polyclonal antibodies,
rabbits were immunized with Uhp1 bands excised from SDS gel, minced in
PBS, mixed with Freund's complete adjuvant (1:1 v/v), and injected
subcutaneously at multiple sites on the rabbit's back. Alternatively,
affinity-purified MBP-Uhp1 fusion protein was injected. Two booster
injections were given with same amount of antigen mixed with Freund's
incomplete adjuvant at weekly intervals. A week after the last
injection, blood samples were collected by puncturing the ear vein, and
specificity of the antibody was established by Western blotting.
Western Blot Analysis and Immunoprecipitation--
Western
blotting was performed by using semi-dry blotter as instructed by the
manufacturer (Sigma). After transfer membrane was blocked with 5% skim
milk in 1× PBS at 4 °C for 2-12 h. Primary antibodies were used at
the following dilutions: 1:250 for anti-Uhp1 and 1:2000 for anti-HA.
Secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG)
was used at 1:2000 dilution. Incubations with antibodies and substrates
were performed as per the manufacturer's instructions (Promega; 20 µl of 50 mg/ml NBT, 10 µl of 50 mg/ml 5-bromo-4-chloro-3-indolyl
phosphate, 40 µl of 1 M MgCl2, 1.5 ml of 1 M Tris·HCl, pH 9.0, in total volume of 10 ml).
Alternatively, horseradish peroxidase-conjugated anti-mouse/anti-rabbit
IgG (1:2000) was used in combination with ECL-Plus (Amersham
Biosciences) detection reagent as per the manufacturer's instructions.
GST Pull-down Assay--
Protein extracts prepared from E. coli cells expressing GST-histone fusion proteins were allowed to
bind to glutathione-Sepharose beads in MTPBS buffer (100 mM
Na2HPO4, 16 mM
NaH2PO4, and 150 mM NaCl) in a
reaction mix of 100 µl containing 1 mg/ml BSA, 50 µl of beads (50%
v/v), and three increasing concentrations of GST-histone fusion protein
for half an hour with shaking. The mixture was washed twice with MTPBS.
To the above reaction mix, 150 µg of protein extract prepared from
h90 p22 Glutaraldehyde Cross-linking Experiment--
Protein extract was
prepared from h90 uhp1 Ni-NTA Chromatography--
Ni-NTA chromatography was performed
as per the manufacturer's instructions (Qiagen). pHis-Ub vector was
transformed into appropriate strains and expressed in the absence of
thiamin as described earlier (50). 40 µg of protein was loaded in the
input lanes, whereas 1 mg of extract was used for binding to Ni-NTA resin.
Chromatin Immunoprecipitation (ChIP) Assay--
Localization of
Uhp1 at mat2 locus was quantitated by ChIP analysis, which
was performed according to Ekwall and Partridge (51), using primers
12799 and 12754 for mat2, and act1For and act1Rev for
act1 gene. PCR conditions are as follows: 94 °C for 5 min; 35 cycles: 94 °C for 40 s, 51 °C for 30 s,
72 °C for 1 min; and 72 °C for 5 min.
Elevated Level of a 22-kDa Protein in sng1-1/rhp6
The 22-kDa protein was purified by conventional methods of fractional
ammonium sulfate precipitation, DEAE-cellulose chromatography, and
electroelution. Purified protein was subjected to microsequencing, but
its N terminus was found to be blocked. Therefore, it was subjected to
digestion with V8 protease, and the major proteolytic fragments were
subjected to microsequencing. Two fragments of 17.5 and 13 kDa were
also found to have their N termini blocked (Fig. 1b).
However, the 7.5-kDa band yielded the sequence KMHAAPKPNYPVVTL, which
upon data base search was found to match completely with an internal
sequence of Obr1 (Fig. 1c) (53). This protein was found
earlier (GenBankTM accession number P30821; see Ref.
53) to be elevated in brefeldin-resistant mutants of S. pombe, although it did not confer brefeldin resistance by itself.
This protein was also reported as a "25-kDa" protein, which was
elevated in crm1 mutant. The crm1 gene was shown
to be involved in the maintenance of higher order chromosomal structure
(54). However, p25 protein did not mediate crm1 function
(54). Expression of p25 gene was found to be
down-regulated by Crm1 indirectly through down-regulation of
transcription factor Pap1 (55). Based on the new functions of
p22/p25/Obr1, as described below, it has been renamed as Uhp1.
Effect of rhp6
To check at what level does sng1-1/rhp6 Overexpression and Deletion of uhp1 Gene Mimic the Mutant Silencing
Defect of rhp6
To assess directly the effect of overexpression and deletion of
uhp1 on silencing, RT-PCR analysis was carried out to
monitor the level of expression of silent copy transcripts in
h90 and Msmto strains, as described
earlier (38). Both overexpression and deletion of uhp1
caused an elevation of mat2Pc transcript only in a switching
h90 strain (Fig. 3,
top panel, lanes 1-3) but not in a non-switching Msmto strain (Fig. 3, top panel, lanes
4-6). (It may be noted that a low level of mat2Pc
transcript is detected even in the vector control of the switching
h90 strain (Fig. 3, top panel, lane
1) but not in non-switching Msmto strain (Fig. 3,
top panel, lane 4). This is in agreement with earlier results (38) where mat2Pc shows a low,
switching-dependent leakiness of expression.) A similar
effect on expression of mat3Mi transcript was observed in
the h90 strain, with overexpression of
uhp1 having a somewhat modest effect (Fig. 3, middle
panel, lane 2) but a stronger effect caused by deletion
of uhp1 gene (Fig. 3, middle panel, lane
3). However, no effect was observed in non-switching strain
Msmto (Fig. 3, middle panel, lanes
4-6). Thus, overexpression of uhp1 elicits a silencing
defect similar to that exhibited by
sng1-1/rhp6
Interestingly, overexpression and deletion of uhp1 gene have
opposite effects on the level of sporulation in the
h90 strain, whereas overexpression causes a
reduction, deletion of uhp1 gene caused an elevation of
sporulation level (Table III). These results suggest that apart from
its effect on silencing, Uhp1 also exerts an inhibitory effect on the
level of switching and/or sporulation. Thus, Uhp1 may also mediate the
role of Rhp6 in switching and/or sporulation. This is consistent with
the fact that rad6/rhp6
To assess the effect of overexpression and deletion of uhp1
on switching, we used the iodine staining assay. Iodine stains a
starchy compound present in spore cell wall. Switching strains produce
cells of both mating types, which mate and sporulate and give dark
staining with iodine, whereas non-switching strains, such as
Msmto, produce no spores and, therefore, give no iodine staining (45). It is particularly interesting that overexpression of
uhp1 in h90 strain produces two types of
colonies, dark staining and light staining (Table III and Fig.
4). Because mating-type region is prone
to rearrangements at a low rate (~10
Microscopic examination showed that the light-staining colony has a
higher level of haploid meiosis as compared with the dark staining
colony (Table III). This level was also higher than that observed in
the h90 strain carrying uhp1 deletion
(Table III), which is surprising in view of equally high levels of
mat2Pc transcript in strains expressing uhp1 on a
high copy vector or having uhp1 deletion (Fig. 3,
lanes 2 and 3). This may be explained by the fact
that RT-PCR data for overexpression of uhp1 represent
predominantly dark colonies (which contain very low, ~1%, of light
colonies), having a low level of haploid meiosis, which is similar to
that observed for uhp1 Ubiquitination of Uhp1 in Vivo and Its Dependence on
Rhp6--
Temperature-sensitive mutants of proteasome subunit Mts3
have been shown to accumulate ubiquitinated derivatives of proteins at
the non-permissive temperature of 36 °C because of lack of degradation of multiubiquitinated proteins by the proteasome (60). To
test directly whether Uhp1 is ubiquitinated in vivo, a
construct expressing His6-tagged ubiquitin (50) was
transformed into wild type, uhp1
To check whether ubiquitination of Uhp1 is cell cycle- and
Rhp6-dependent, we subjected wild type and
rhp6 Uhp1 Is Associated Transiently with Silent Locus mat2P during S
Phase--
The association of Uhp1 with silent locus mat2P
was checked by ChIP assay at 0, 2, and 4 h after release of HU
arrest in a wild type strain expressing pHA-Uhp1. We find that Uhp1 is
maximally associated with mat2P at 2 h, which coincides
with S phase but not at 0 and 4 h (Fig.
7). Thus, Uhp1 appears to be associated with silent locus mat2P only during S phase.
Uhp1 Contains the Histone-fold Motif Similar to Histone H2A and
Interacts with Histone H2B--
Motif search did not reveal any
structural motif or nuclear localization sequence in Uhp1, and only one
short sequence with sub-optimum similarity to the PEST motif for
ubiquitination was found (61, 62). However, a direct comparison with
histone-fold motifs found in core histones by ClustalW analysis
revealed a close similarity of residues 13-76 in Uhp1 to the
histone-fold motif in histone H2A, with about 20% identity and 40%
similarity (Fig. 8a). This
motif has been identified not only in core histones (63-65) but also
in some TBP-associated factors (66). Maximum similarity was
found in the middle helix
To check directly whether Uhp1 can interact with its cognate partner
H2B, like histone H2A, we performed GST pull-down assay by incubating
the extracts prepared from wild type strains expressing the HA-tagged
uhp1 gene with GST histone fusion proteins, where GST was
fused with histones H2A, H2B, and H3. Western blotting with anti-GST
antibody confirmed the expression of fusion proteins from three
GST-histone recombinant clones (Fig.
9a, lanes
1-3, arrowheads). Results indicate that histone H2B
exhibits a strong interaction with Uhp1, whereas histone H2A does not
interact at all, and H3 interacts relatively weakly (Fig.
9b, compare lanes 7-9 with lanes 4-6
and 10-12). Thus, Uhp1 does interact with the cognate
histone partner of H2A. Interaction with histone H4 could not be
checked because the GST-H4 fusion construct could not be expressed in
E. coli, presumably because of unsuitable codon bias.
We also checked whether Uhp1 can interact with itself and form
homodimers or multimers. Results of glutaraldehyde cross-linking show
that Uhp1 does from homodimers in vitro (Fig.
9c). However, no multimers could be detected.
Uhp1 Derivatives Lacking Histone-fold and A Novel Mechanism for Propagation of Chromatin State--
The main
objective of this study was to identify the proposed mediator of Rhp6
in silencing. In an earlier study, Rhp6p was hypothesized to play a
role in coupling the assembly of chromatin to DNA replication
associated with switching (38, 39). Accordingly, existence of a
mediator of Rhp6, which participates transiently in chromatin assembly
at switching mating-type loci, was proposed. In addressing this issue,
we have made the following main findings. First, we find that a
protein, renamed here as Uhp1, which contains the histone-fold motif
and acts like H2A in binding to its cognate partner H2B, is elevated in
the sng1-1/rhp6
It remains to be checked how Uhp1 may be involved in chromatin
assembly. It may be speculated that either one or both forms (ubiquitinated and un-ubiquitinated) of Uhp1 may associate with nascent
nucleosomes, where they may substitute for H2A and/or H2B, generating a
unique metastable nucleosome structure at the replication fork. This
structure may facilitate proper assembly of inactive chromatin after
the proteasome pathway helps to channel the ubiquitinated Uhp1 for
degradation concomitantly with recruitment of histone H2A. In contrast,
the continuous presence of Uhp1, as happens in
sng1-1/rhp6 Mechanism Governing Regulation of Uhp1--
Surprisingly,
rhp6
The transient localization of Uhp1 at mat2P raises the
question of how it may be transported to the nucleus and what regulates the association. Nuclear localization is surprising in view of the
absence of the nuclear localization sequence. It is possible that
nuclear entry may be regulated either by ubiquitination itself or by an
unknown protein that facilitates its nuclear entry. The S
phase-specific association with mat2 locus may be dictated
by a previously undetectable modification, like phosphorylation. Another intriguing observation is the lack of any ubiquitination motif
in Uhp1. Although not all proteins that are ubiquitinated possess such
motifs, it is possible that ubiquitination of Uhp1 may be facilitated
by ubiquitin-protein isopeptide ligase activity, which remains
to be discovered (61).
Epigenetic Function of Uhp1 in Switching and
Silencing--
Recently, histone-code hypothesis has been proposed to
explain the epigenetic phenomena wherein heterochromatin and
euchromatin regions are associated with differently modified histones
(71). According to this hypothesis, specifically modified histones may present a code for recognition by specific proteins, either
transcription co-factors or repressors, which lead to either gene
expression or gene silencing, respectively. Our study, showing the role
of ubiquitination of a histone-like protein in chromatin assembly and
in propagation of heterochromatin structure, helps to extend the
histone-code hypothesis to non-histone chromosomal proteins. An
interesting scenario may be where ubiquitinated Uhp1 is recognized by
one or more of heterochromatin-associated proteins, like Clr4 (see
below). In this context, an interesting effect observed when Uhp1 was
overexpressed in h90 strain was manifestation of two
alternative iodine staining colonies, the dark staining and the light
staining. The light staining colonies exhibit a persistence of the
light staining even after the plasmid was lost, suggesting a role of
Uhp1 in establishing a stable epigenetic state.2 Thus, Uhp1
may be important for initiating assembly of a structure that can
propagate itself. This finding is reminiscent of the results of Grewal
and Klar (30), where deletion of the K region spanning the
mat2-mat3 interval generated two alternative epigenetic states showing different extents of switching/silencing. These states
were metastable and switched to the other state at a low rate
(~10 Interaction of Uhp1 with Other Factors Involved in Switching and
Silencing--
To check the genetic interactions with other silencing
factors, we transformed Uhp1 into swi6 and
clr1-clr4 mutant strains. Interestingly, we observed the
effect of overexpression of Uhp1 only in the
h90, clr4 mutant, which yielded a light
staining phenotype with reduced sporulation and switching but an
increase in the level of haploid meiosis. (The mating-type organization
and the level of double strand break are not affected in these
transformants.) Interestingly, unlike h90 strain,
where less than 1% transformants gave light staining, almost 25% of
transformants of the chromodomain mutant W31G and 2% in the case of
SET domain mutant G486D of Clr4 (25) gave light staining, suggesting
that wild type Clr4 may function in conjunction with Uhp1. Recently,
SET domain of Clr4 has been shown to be associated with histone
methyltransferase activity specific for Lys-9 in histone H3 (25, 26).
Furthermore, Lys-9-methylated histone H3 is bound to Swi6 in
heterochromatin regions (26, 27). It may be interesting to check
whether Uhp1 physically interacts with Clr4 in vivo and
helps to recruit it to heterochromatin regions.
Does Uhp1 Function Like a TAF?--
Studies on
TBP-associated factors (TAFs) in Drosophila and human
have led to the identification of TAFs that are homologous in their
histone-fold regions to H2B, H3, and H4. However, no TAF showing
homology to histone H2A has been identified (65, 66). Because of its
similarity to histone H2A in histone-fold region and its interaction
with H2B, the cognate partner of histone H2A, it is possible that Uhp1
may be the missing TAF corresponding to histone H2A. Here it is
pertinent to note the effect of overexpression of Uhp1 derivatives
lacking the histone-fold and Possible Conservation of Uhp1 Function--
Finding of a high
level of homology to trpRBP in several bacterial species is surprising,
presenting the possibility of a horizontal transmission between
bacteria and yeast. It is tempting to speculate that a certain class of
transcriptional repressors may have crossed the species barrier.
Furthermore, the existence of a protein of unknown function but similar
size from S. cerevisiae with a high level of homology to
Uhp1 is quite intriguing. It would be interesting to check whether it
performs a similar function, i.e. whether it serves as a
target of Rad6 and plays a role in silencing in the budding yeast.
These questions will be addressed in future studies.
mutant and
rhp6
as compared with wild type strain. Both the deletion and overexpression of the gene encoding this protein elicit
switching-dependent loss of silencing. Furthermore, the 22-kDa protein undergoes Rhp6-dependent multiubiquitination
and associates with mat2 locus during S phase in wild type
cells. Interestingly, it contains a histone-fold motif similar to that of histone H2A, and like histone H2A, it interacts strongly with histone H2B in vitro. These results indicate that the
22-kDa protein, renamed as the ubiquitinated histone-like protein Uhp1,
is an in vivo target/mediator of Rhp6 in silencing. Thus,
regulation of association of Uhp1 with chromatin and ubiquitination
followed by degradation may play a role in reestablishment of inactive chromatin structure at the silent mating-type loci.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
has been shown to play a role in
establishment of epigenetic chromosomal state, which is stably
propagated during mitosis and meiosis (35), through interaction with
the chromodomain protein Swi6. Thus, pol
may play a role in
silencing presumably through recruitment of Swi6 (35, 36), indicating
that DNA replication is directly coupled to the assembly of silent
chromatin structure in S. pombe (36, 37).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
List of yeast strains and plasmids used in this study
-mercaptoethanol). Then twice the volume of Buffer B (Buffer A + 0.01 mM
phenylmethylsulfonyl fluoride) was added to the pellet. In addition,
protease inhibitors (20 µg each of pepstatin, aprotinin, and
leupeptin) and an equal volume of glass beads (425-600 µm
diameter) were added. Cells were broken either by vortexing at 4 °C
or in a homogenizer. Each 1-min cycle was followed by a 30-s incubation
on ice until 80% lysis occurred (lysis of cells was checked under the
microscope by adding 1% SDS). The lysed cells were centrifuged at
55,000 rpm for 30 min at 4 °C in TL-100-3 rotor of TL-100 Ultra
centrifuge (BD Biosciences) or SW-41 rotor of BD Biosciences at 30,000 rpm for 45 min. Supernatant was collected, aliquoted, and frozen at
70 °C. Protein concentration was estimated according to Bradford (46) using BSA as standard. SDS-PAGE was carried out essentially according to Laemmli (47).
mutant,
it was purified by conventional methods from the mutant strain. Total
cell extract was prepared from a 4-liter culture of mutant strain grown
in YEA medium at 30 °C. Fractional ammonium sulfate precipitation
was done at 4 °C, and the 22-kDa protein, which was referred to as
p22, was found to precipitate maximally at 40-60% ammonium sulfate
concentration range. The precipitated fraction was redissolved,
dialyzed, and subjected to DEAE-Sepharose chromatography, where p22
appeared in the flow-through fraction. Finally, the flow-through
fraction was subjected to preparative SDS-PAGE; the p22 bands were
excised and electroeluted. Upon SDS-PAGE and silver staining the
protein was found to be purified to apparent homogeneity.
Primers used in this study
transcripts
have been described earlier (38). Whereas RT-PCR products of
mat2Pc and mat3Mi transcripts were detected by
Southern blotting that for pol
could be detected by
ethidium staining, which may reflect relative abundance of their
respective transcripts. Northern blotting and hybridization were
performed according to Sambrook (49), and the radioactive bands were
quantitated on Bio-Rad Molecular Imager Fx using Quantity One version
4.2.2 software.
-D-galactopyranoside was added to a
final concentration of 1 mM when the
A600 reached 0.4-0.5. Cultures were
incubated further at 37 °C for 3-6 h. Cells were collected by
centrifugation and suspended in either PBS or TE containing 1 mM phenylmethylsulfonyl fluoride and sonicated 3-4 times
at a frequency of 15-20 for 20 s each on Branson sonicator. After sonication 0.1% Triton X-100 was added, and the protein extract was
collected by centrifugation at 4 °C for 15 min. Aliquots of protein
extracts were frozen at
20 °C.
rhp6
strain
expressing pHA-Uhp1 construct, 1 mg/ml BSA, and 1× binding buffer (5×
Binding Buffer 750 mM NaCl, 100 mM Tris·Cl,
pH 8.0, 5 mM EDTA, 0.5% Triton X-100, and 5 mM dithiothreitol) were added. The reaction mixture was
incubated at room temperature for 1 h and washed 4 times with
washing buffer (100 mM NaCl, 20 mM Tris·Cl, 1 mM EDTA, 0.1% Triton X-100, mM EDTA).
After adding 1× SDS loading dye, samples were boiled for 10 min and
immunoblotted with anti-HA antibody.
strain
expressing the plasmid pHA-Uhp1 and subjected to cross-linking at room
temperature for 30 min in a reaction mixture (50 µl) containing 20 mM Tris·HCl, pH 7.5, 1 mM ZnCl2,
35 µg of HA-Uhp1 extract, and 0.005 and 0.01% glutaraldehyde. The
reaction was stopped by addition of SDS loading buffer. Samples were
boiled and immunoblotted with anti-HA antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Mutant and rhp6
Strains--
Earlier, we hypothesized that a
protein target of Rhp6 may participate transiently in chromatin
remodeling at the silent loci during switching; thereafter it is
ubiquitinated, channeled to the proteasome pathway, and degraded (38,
39). Such a protein may be present at a low level in steady state
population of wild type cells. However, in
sng1-1/rhp6
mutant it may
accumulate because of lack of ubiquitination and degradation, and its
continued association may perturb chromatin structure leading to
derepression of silent cassettes (38, 39). To test this idea, we
compared electrophoretic patterns of proteins from wild type and
rhp6
/sng1-1 mutant strains grown at
30 and 36 °C. The level of a 22-kDa protein was found to be greatly
elevated at 30 °C in the
sng1-1/rhp6
mutant as compared with
wild type strain (Fig. 1a,
compare 1st and 3rd lanes). An elevated level of
the 22-kDa protein was also observed in the rhp6
strain.2 Surprisingly,
however, the level of elevation was reduced at 36 °C as compared
with 30 °C in rhp6
mutant (Fig.
1a, compare 3rd and 4th lanes). The
reason is not clear. However, it is possible that some other
ubiquitination pathway, like Hus5 (52), may be activated at 36 °C,
which partially reduces the p22 level.
View larger version (50K):
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Fig. 1.
A protein of 22 kDa, elevated in
sng1-1/rhp6 mutant as
compared with wild type cells, is identical to the previously reported
proteins p25 and Obr1. a, protein extracts were prepared
from wild type (WT) and sng1-1/rhp6
mutant cells, which were grown at 30 and 36 °C. 30 µg of each
sample was subjected to SDS-PAGE (12.5%) and gel stained with
Coomassie Blue. M indicates molecular weight marker.
b, V8 protease digestion pattern of p22. 20 µg of purified
p22 was subjected to V8 protease digestion and a small aliquot
subjected to SDS-PAGE (12.5%) followed by silver staining.
M, molecular weight marker. c, amino acid
sequence of p22/p25/Obr1. Upon microsequencing, 7.5-kDa internal
polypeptide shown in b yielded the 15-amino acid sequence
(underlined), whereas the intact p22/Uhp1 as well as the
17.5- and 13-kDa bands were found to have their N termini blocked.
BLAST search revealed the protein to beidentical to the
previously reported p25 protein (53) and Obr1 (51).
/sng1-1 Mutation on uhp1 mRNA and
Protein--
We obtained "p25" gene described earlier
(55), and the strain carrying disruption of the p25
gene from Dr. Toda. Western blotting using antibody raised against
p22/Uhp1 protein showed absence of p22/Uhp1 band in p25
deletion strain, and enhanced levels in a strain carrying
p25/uhp1 gene on a high copy plasmid,2 thus
confirming that p22/uhp1 was identical to the product of obr1/p25 genes described earlier (53, 54).
mutation affect Uhp1 expression, we carried out Northern and Western
blot analysis for wild type and sng1-1/rhp6
mutant strains. Northern blots were probed with uhp1 and
cdc2 genes (Fig.
2a, lanes 1 and
2). PhosphorImager analysis showed that
sng1-1/rhp6
mutation causes a reproducible
2-fold elevation of the uhp1 mRNA, whereas Western
blotting results showed an overall 16-fold higher Uhp1 level in
sng1-1/rhp6
mutant as compared with wild type
strain (Fig. 2b, compare lanes 2 and
1; the faint band in the wild type lane 2 is
indicated by an arrowhead). Thus,
sng1-1/rhp6
mutation affects the expression of
uhp1 gene modestly at a transcriptional level but more
strongly at a post-transcriptional level (about ~8-fold).
View larger version (26K):
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Fig. 2.
Effect of
sng1-1/rhp6 mutation on the level of
uhp1 mRNA and protein. a, RNA was
prepared from wild type (wt) (lane 2) and
sng1-1/rhp6
mutant (lane 1) strain.
20 µg of total RNA was subjected to Northern blotting and
hybridization with radiolabeled uhp1 (upper
panel) or cdc2 (lower panel) probes. Signal
was recorded and quantitated using Bio-Rad PhosphorImager.
b, protein extracts prepared from wild type (lane
2) and sng1-1/rhp6
strain (lane
1) were subjected to immunoblotting with anti-Uhp1 (upper
panel) and TAT1 (lower panel) antibodies. Signals were
quantitated on Gel Doc system of Amersham Biosciences. The faint band
corresponding to Uhp1 in the wild type extract is indicated by an
arrowhead.
--
To check the involvement of Uhp1 in
silencing, uhp1 gene cloned on a high copy vector was
expressed in the both homothallic (h90) and
heterothallic nonswitching strain Msmto (this strain carries a deletion of cis-acting sequences flanking the mat1 locus
that are required for generation of double strand break and hence
switching; see Ref. 56). Likewise, disruption strains were generated in both switching and nonswitching backgrounds, and their phenotype was
compared with vector controls. Interestingly, we find that both
overexpression and deletion of Uhp1 elicit haploid meiosis phenotype,
indicative of the silencing defect (Table
III). However, this effect was observed
only in the switching (h90) strain and not in the
non-switching (Msmto) strain (Table III). Thus,
manifestation of defects caused by overexpression and deletion of
uhp1 show a dependence on switching competence of
strains, which is similar to the phenotype displayed by
sng1-1/rhp6
mutant (38).
Changes in the level of zygotic asci and haploid meiosis (hm) asci due
to overexpression and deletion of the uhp1 gene
denotes overexpression;
denotes deletion. For each measurement
500 cells were counted.
mutant (38), i.e. the
effect is switching-dependent. These results show that Uhp1
does function as an in vivo mediator of Rhp6.
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Fig. 3.
Both overexpression and deletion of
uhp1 gene cause a loss of silencing, which is
switching-dependent. RT-PCR was carried out for
mat2Pc (top panel), mat3Mi
(middle panel), and pol (bottom
panel) transcripts using RNA prepared from wild type
h90 (lanes 1-3) or Msmto
strain (lanes 4-6), either alone (lanes 1 and
4) or containing the uhp1 gene on a high copy
vector pYA292 (lanes 2 and 5; a kind gift of T. Toda) or carrying uhp1 deletion (lanes 3 and
6).
mutants have reduced
levels of sporulation (57, 58).
4/generation) (59),
some of which may possibly affect silencing, it is possible that cells
of the light colony may have undergone a rearrangement. Such
rearrangements are known to occur more frequently in the
h90 as compared with the Msmto strains
(59). However, Southern analysis showed no such rearrangement in the
light staining colonies.2 Interestingly, light staining
colonies again produced colonies that give light staining and switched
to dark staining at a low rate of
~10
4/generation.2 Thus, dark and light
staining colonies may represent alternative metastable epigenetic
states of switching and/or silencing, like those exhibited by the
strain carrying deletion of K region, as reported earlier (30).
View larger version (40K):
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Fig. 4.
Overexpression reduces whereas deletion of
uhp1 enhances the iodine staining of
h90 strain but not Msmto
strain. h90 (upper panel) and
Msmto (lower panel) strains, transformed with
vector alone (left panels) or uhp1 gene on a high
copy vector pYA292 (middle panel) or carrying
uhp1 deletion (right panel), were streaked
on PMA plates lacking leucine. Colonies were stained with iodine after
growth for 4 days and photographed. D and L
represent colonies that give dark and light staining,
respectively.
strain (Table III).
, and mts3-1
strains. Cultures of transformed strains were first grown at 25 °C
and then shifted to 36 °C for 4 h. Extracts prepared from these
strains were subjected to Ni-NTA chromatography followed by
immunoblotting with anti-Uhp1 antibody. The presence of bands with
reduced mobility in mts3-1 mutant (Fig.
5, lane 1) and their absence
in uhp1
strain (Fig. 5, lane 2) directly demonstrate that Uhp1 undergoes multiple ubiquitination in
vivo. Fainter bands are also observed in wild type cells (Fig. 5,
lane 3), which may have reduced levels of ubiquitinated
Uhp1, as compared with mts3-1 mutant, because of a
functional proteasome.
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Fig. 5.
Uhp1 is ubiquitinated in
vivo. Accumulation of ubiquitinated forms of Uhp1 in
proteasome mutant mts3-1 under non-permissive conditions.
Wild type (wt) (lane 3), uhp1
(lane 2), and mts3-1 mutant (lane 1)
strains carrying the His-Ub vector were grown initially at 25 °C
under conditions that allow expression of His6-tagged
ubiquitin. Cultures at an A600 of 0.2-0.5 were
shifted to 36 °C and grown for 4 h. Protein extracts were
prepared, and 1 mg of each protein sample was subjected to Ni-NTA
chromatography. The Ni-NTA-bound fractions were immunoblotted with
anti-Uhp1 antibody.
mutant strains expressing His-Ub vector
to cell cycle arrest in S phase with hydroxyurea. Cells were harvested
at hourly intervals after release of HU arrest, and the level of
ubiquitination of Uhp1 was monitored after Ni-NTA chromatography,
followed by immunoblotting with anti-Uhp1 antibody. Results shown in
Fig. 6 indicate the appearance of one
major ubiquitinated band of Uhp1 in wild type cells at 0 h (Fig.
6a, lower panel, lane 5), whose level
shows a slight (~2-fold) increase along with appearance of additional fainter bands with reduced mobility at 2 (corresponding to S phase) and
4 h (Fig. 6a, lower panel, lanes
6-8). Importantly, no such bands were observed in the
rhp6
mutant (Fig. 6b, lower
panel, lanes 5-8). These results clearly demonstrate
that Rhp6 is essential for ubiquitination of Uhp1.
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Fig. 6.
Rhp6 dependence of ubiquitination of
Uhp1. Cells of wild type (WT) (a) and
rhp6 /sng1-1 mutant (b)
strain expressing His-Ub vector were first subjected to HU arrest for
4 h. Cells were washed free of HU and grown further for 0-4 h.
Septation index was monitored and plotted (a and
b, top panels). a and
b, lower panels, 1.0 mg of protein extract
prepared from samples harvested at 0 (lane 5), 1 (lane
6), 2 (lane 7) and 4 h (lane 8) was
subjected to Ni-NTA chromatography, and the Ni-NTA bound fractions were
electrophoresed along with 40 µg of each protein fraction at 0 (lane 1), 1 (lane 2), 2 (lane 3), and
4 h (lane 4) and immunoblotted with anti-Uhp1
antibody.
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Fig. 7.
Uhp1 is localized to mat2
locus during S phase. ChIP analysis of a wild type
h90 strain expressing an integrated copy of
HA-tagged Uhp1. Cells were grown to early log phase
(A600 of 0.2-0.3) and subjected to HU arrest.
Samples were collected at 0 (lane 1), 2 (lane 2),
and 4 h (lane 3) after release of HU arrest and
subjected to ChIP analysis using oligonucleotides to amplify
mat2P and act1 genes. The
mat2-to-act1 signal ratio was determined for each
NIP and IP sample, and the enrichment ratio was determined using
the IP/NIP ratio for 0 h sample as 1. NIP, control
sample, non-immunoprecipitated; IP,
immunoprecipitated.
2 and loop regions L1 and L2 (Fig.
8a; Ref. 65). However, key basic residues that are required
for interaction with DNA in H2A are not present in Uhp1 (Fig.
8a; Ref. 65). (Slightly less similarity was detected toward
histone H2B and none with histones H3 and H4.2)
Intriguingly, BLAST search (67) also revealed a strong homology with
trp repressor binding protein (trpRBP) in E. coli and
Bacillus subtilis (E-value = 1e
28; Fig. 8c). It has been shown that
trpRBP does not interact with DNA but interacts with and modulates the
activity of trp repressor (68). Thus, like histone H2A, Uhp1 could
interact with either histone H2B and/or with other proteins that
regulate mating-type silencing. Interestingly, BLAST search also showed
a high level of homology toward a hypothetical protein of S. cerevisiae (YDR032c, GenBankTM accession number
S61585; E-value = 3e
49) with 60%
identity and 77% similarity (Fig. 8b). It is possible that
this protein may be the in vivo target of Rad6p in S. cerevisiae. Interestingly, both these proteins are similar in size
to Uhp1 and share homology with it all along its length including the histone-fold.
View larger version (43K):
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Fig. 8.
Uhp1 contains histone-fold motif similar to
that of histone H2A and presence of homologous proteins in E. coli and S. cerevisiae. a,
ClustalW alignment of Uhp1 with histone-fold motif of histone H2A.
Maximum homology is seen in central helix 2. Residues in H2A that
have been shown to interact with DNA are also indicated (65), which are
lacking in Uhp1. b and c, BLAST search also shows
a high degree of homology of Uhp1 with a hypothetical protein of
unknown function (YDR032c) from S. cerevisiae (b)
and with trp repressor-binding protein (trpRBP) from
E. coli (c).
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Fig. 9.
Uhp1 exhibits specific interaction with
histone H2B in vitro. a, extracts
prepared from E. coli strains expressing GST-H2A (lane
1), GST-H2B (lane 2), GST-H3 (lane 3), and
GST alone (lane 4) were immunoblotted with anti-GST
antibody. Arrowheads indicate the positions of GST-histone
fusion proteins. b, GST pull-down assay was performed by
adding nothing (lane 1), beads alone (lane 2),
GST extract (lane 3), and three increasing amounts of
extracts of cells expressing GST-H2A (lanes 4-6), GST-H2B
(lanes 7-9), and GST-H3 (lanes 10-12) to the
extract prepared from S. pombe strain lacking
uhp1 gene but containing the pHA-uhp1 construct.
Proteins remaining bound to glutathione-agarose beads were
immunoblotted with anti-HA antibody. c, Uhp1 can form
homodimers in vitro. Extract prepared from a wild type
strain expressing pHA-Uhp1 was subjected to cross-linking with 0.005 and 0.01% glutaraldehyde and subjected to immunoblotting with anti-HA
antibody. 0 denotes no glutaraldehyde.
2 Helix Exert a
Dominant Negative Effect on Switching--
To check whether the
histone-fold motif and the central
2 helix play a critical role in
the function of Uhp1, we constructed clones of Uhp1 lacking
histone-fold motif and
2 helix, and expressed them in the
h90 strain. Interestingly, all the transformants
carrying the constructs lacking histone-fold or
2 helix gave light
staining with iodine (Fig. 10).
Southern analysis showed that these transformants had normal
mating-type organization and the level of double strand break.2 Microscopic examination of light staining
transformants showed a greatly reduced level of sporulation (Table
IV). This defect may be due either to
reduced mating efficiency caused indirectly by misregulation of
mat1 transcription or due to defects in switching and/or
sporulation. Thus, constructs lacking histone-fold or
2 helix exert
a dominant negative effect either on mating, switching, or
sporulation.
View larger version (60K):
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Fig. 10.
Histone-fold motif is important for Uhp1
function. h90 strain carrying a
mat3-linked ura4 marker was transformed with vector alone,
uhp1 gene on high copy vector (pYA292; Table I), and
uhp1 derivative clones lacking histone-fold motif or 2
helix. Transformants were streaked on PMA plates lacking leucine. After
4 days of growth colonies were stained with iodine and
photographed.
Level of zygotic asci and haploid meiosis (hm) asci on overexpression
of Uhp1 derivatives lacking histone-fold and 2 helix
denotes Nil. For each measurement 500 cells were counted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant. Second, Uhp1 is
regulated predominantly at post-translational level by
Rhp6-dependent ubiquitination. Third, Uhp1 associates with
chromatin in a cell cycle-dependent manner. Finally, both overexpression and the absence of Uhp1 elicit a silencing defect in
wild type strains. These findings suggest a unique mechanism of
propagation of chromatin state: by transient association of Uhp1 with
chromatin, which is regulated by ubiquitin-mediated turnover. The
modified as well as unmodified forms of Uhp1 may associate with
chromatin during S phase, where after Uhp1 is degraded. Their
association with silent mat2P donor locus during S phase coincides with switching, which occurs by a replication-coupled recombination mechanism (69). Thus, Uhp1 may participate in chromatin
assembly during replication of switching mating-type donor loci. Such a
role is consistent with the proposed function of Rhp6 in
reestablishment of chromatin structure of switching donor loci
(38).
mutant or when uhp1 is
present on a high copy vector, might lead to a persistent alteration in
chromatin structure, which may cause derepression of the silent loci.
It is possible that in rhp6
mutant, an excess
amount of Uhp1 protein may result in an altered nucleosome structure
with Uhp1 in place of H2A, which may not be able to form
heterochromatin structure at the switching donor loci. Alternatively,
chromatin lacking in ubiquitinated Uhp1 may not be recognized by other
heterochromatin factors like Clr4 (see below). A similar
ubiquitin-mediated regulatory mechanism operates in cell cycle control
where cyclin degradation by ubiquitin-mediated proteolysis is critical
for G1-S transition (70). Thus, a unique feature of this
study is to demonstrate that a similar regulatory mechanism may operate
in facilitating the propagation of chromatin structure.
mutant shows a 2-fold increase in
uhp1 mRNA, as a role of Rhp6 in transcription, is quite
unexpected. It is possible that Rhp6 may affect the stability of a
specific regulator of expression of Uhp1, like Pap1 or Crm1 (55).
However, as expected, our results suggest that Uhp1 level is regulated
predominantly at the post-translational level. In addition, our results
indicate that Rhp6 is the ubiquitin-conjugating protein activity
responsible for the ubiquitination of Uhp1, because no ubiquitination
was detected in rhp6
mutant, whereas multiply
ubiquitinated moieties were detected in wild type cells, which
accumulate further in mts3-1 mutant.
4/generation) during mitotic growth. Interestingly,
they also behaved as Mendelian alleles during meiosis, indicating that
they had an imprinted memory of the chromatin structure, which could be propagated during mitosis and meiosis (30).
2 helix; most of the transformants
exhibit a much reduced iodine staining with a lower level of switching
and a defect in silencing with a high level of haploid meiosis. On the
other hand, overexpression of intact Uhp1 produces light staining
colonies at a rate of ~1%. Thus, the histone-fold region may play an
important role in the assembly of the functional complex involved in
switching and silencing.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to A. Klar, P. Nurse, M. Yanagida, T. Toda, and S. Moreno for plasmids and strains. We thank G. Sahni for microsequencing. We are particularly thankful to S. Ahmed, S. Arora, and R. Kumar for technical assistance, T. Toda for critically reading the manuscript, L. Iveleen for editorial assistance, and an anonymous reviewer for constructive suggestions for improvement of the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to the work.
¶ To whom correspondence should be addressed: Institute of Microbial Technology, Sector 39A, Chandigarh-160036, India. Tel.: 91-172-695215 (ext. 443); Fax: 91-172-690585/690632; E-mail: jag@imtech.res.in.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M212732200
2 S. Saini, A. Naresh, and J. Singh, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SIR, silent
information regulator;
Clr, cryptic loci regulator;
Swi, switching;
Rhp6, Rad6 homologue in pombe;
sng, silencing not governed;
PMA, pombe
minimal medium with adenine;
HU, hydroxyurea;
HA, hemagglutinin;
Ub, ubiquitin;
pol, DNA polymerase
;
TAF, TBP-associated
factor;
ChIP, chromatin immunoprecipitation;
Ni-NTA, nickel-nitrilotriacetic acid;
HA, hemagglutinin;
RT, reverse
transcription;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
GST, glutathione S-transferase;
CAPS, 3-(cyclohexylamino)propanesulfonic acid;
TBP, TATA-binding
protein.
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REFERENCES |
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