Chromatin remodeling is required for efficient transcription of eukaryotic genes. In a genetic selection for budding yeast mutants that were defective in induction of the phosphate-responsive PHO5 gene, we identified mutations inARG82/IPK2, which encodes a nuclear inositol polyphosphate kinase. In arg82 mutant strains, remodeling ofPHO5 promoter chromatin is impaired, and the adenosine triphosphate–dependent chromatin-remodeling complexes SWI/SNF and INO80 are not efficiently recruited to phosphate-responsive promoters. These results suggest a role for the small molecule inositol polyphosphate in the regulation of chromatin remodeling and transcription

Haswell, Elizabeth S.

Miller, Aimee L.

O'Shea, Erin K.

Steger, David J.

Wente, Susan R.

Caltech Authors - Main

Supporting online text
1. To isolate mutant strains defective in the activation of PHO5 transcription, we
treated the parental strain [EY696 (K699 MATa pho80ts can1::pPHO5-CAN1
pho81∆::TRP1B ade2-1 trp1-1 leu2-3, 112 his3-11, 15 ura3-1)] with ethylmethane
sulfonate to ~50% viability and plated it for growth on SD - arginine + 8 µg/ml
canavanine medium at 37°C.  Cells with pPHO5-CAN1, a PHO5 promoter-CAN1 fusion
construct, are sensitive to canavanine in conditions that induce PHO5 expression.  We
isolated 20 mutants carrying recessive mutations affecting Pho5 acid phosphatase
activity.  Ten of these were divided into four complementation groups, with the
remaining ten unassigned.  Four mutants with mutations in PSE1 or ARG82 displayed
defects in PHO5 transcription.  The remaining 16 mutants were found to contain defects
in Pho5 activity that are downstream of transcription, and were not examined further.
2. The arg82-153 allele was recovered by gap repair of two different gapped
vectors.  The rescued allele from each vector was determined to be identical in the
ARG82 coding region by sequencing.  arg82-153 contained a single C to T substitution at
position 541, introducing a stop codon following the inositol-binding motif, and failed to
rescue the PHO5 uninducible phenotype of arg82∆ cells.  Since the arg82-153 allele
rescued the temperature-sensitive lethality of the arg82∆ strain (2, 5), it is unlikely to be
a null.
3. PHO5 and PHO84 expression is partially constitutive in the following strains,
grown in high phosphate conditions: plc1∆ pho80ts, arg82∆ pho80ts, and kcs1∆ pho80ts
(2).  This partially constitutive expression is dependent on PHO81, suggesting that
inositol polyphosphates may play a role in the phosphate signaling pathway upstream of
MS# 1078062, Supplementary Online Material 2
the CDK inhibitor Pho81.  This additional role for inositol polyphosphates may also be
the cause of increased binding of Ino80, Snf2, and Pho4 to the PHO5 and PHO84
promoters in the arg82∆ strain (Figs. 2c, 2d, 3a, and S4, DMSO WT  vs. arg82∆), as
these experiments were carried out using a strain that is PHO81+.
4. Snf2 and Ino80 are the catalytic subunits of the SWI/SNF and INO80 remodeling
complexes, respectively.  Because ino80∆ and snf2∆ leu2-3 trp1-1 strains are not viable
in K699 yeast, we deleted nonessential components that impair function of the
remodeling complexes.
5. We use the Pho85F82G strain to control transcription of phosphate-responsive
genes because addition of 1-NaPP1 to cells inhibits Pho85 function within minutes (7)
and is a more selective perturbation of the pathway than shifting pho80ts cells to 37°C.
Like the arg82∆ pho81∆ pho80ts strain, arg82∆ Pho85F82G cells are also defective for
PHO5 transcription and chromatin remodeling (2).
6. We find that strains with mutations in SNF6 and SNF2 induce PHO5 transcription
with slower kinetics when depleted for phosphate (2).  A defect in PHO5 transcription
induction in no phosphate medium has been previously reported for an ino80∆ strain (8).
In general, mutations in subunits of the SWI/SNF and INO80 complexes impair PHO5
induction to a lesser degree when cells are depleted of phosphate versus treated with 1-
NaPP1 in high phosphate (2).  This is consistent with an earlier report (9) proposing that
an additional signal(s), independent of Pho80-Pho85, contributes to activation of PHO5
transcription when cells are starved for phosphate.
7. Gcn5- and Esa1-containing histone acetyltransferase (HAT) complexes acetylate
histones H3 and H4 at the PHO5 promoter and regulate its transcription (10-12).  We did
MS# 1078062, Supplementary Online Material 3
not observe detectable changes in the acetylation levels of histones H3 or H4 at the
PHO5 promoter in arg82∆ cells, suggesting that Gcn5 and Esa1 function may not be
influenced by inositol polyphosphates (2).
Supporting figures
Figure S1.  The arg82-153 mutant is defective in PHO5 mRNA induction and chromatin
remodeling.  To control induction of the PHO5 gene, which encodes a secreted acid
phosphatase, a strain carrying a temperature sensitive allele of PHO80 was used (1).  In
Figure S1
WT
25
Cla I
protected
accessible
- - - -
37 25 37
arg82-153
oC
PHO5
ACT1
PHO84
WT
arg82-
153
oC 25 2537 37
A
B
% cut17 63 13 20
MS# 1078062, Supplementary Online Material 4
the pho80ts strain, PHO5 is repressed at the permissive temperature (25°C) and is induced
when shifted to the restrictive temperature (37°C).  (A) Northern analysis of pho81∆
pho80ts (WT) and arg82-153 pho81∆ pho80ts strains grown under repressing conditions
(25°C) or shifted to inducing conditions (37°C) for one hour.  For all experiments, strains
were grown in synthetic high phosphate medium and PHO5 and PHO84 RNA levels
were quantified by normalizing to ACT1.  The mRNA level in the mutant was 16-fold
lower than that in the wild-type strain under inducing conditions.  Not all Pho4-
dependent, phosphate-responsive genes are affected by the arg82-153 allele to the same
degree; PHO84, encoding an inorganic phosphate transporter, was induced to 80% of
wild-type levels, suggesting that Pho4 can activate transcription in the arg82-153 strain.
Consistent with a model in which ARG82 functions downstream of PHO4, the subcellular
localization of Pho4 is appropriately regulated in the arg82-153 mutant (2).  (B) To
determine if the transcriptional defect in the arg82-153 strain is the result of a block in
chromatin remodeling, we examined Cla I restriction enzyme accessibility for pho81∆
pho80ts (WT) and arg82-153 pho81∆ pho80ts strains grown under repressing conditions
(25°C) or shifted to inducing conditions (37°C) for one hour.  The assay was performed
as described elsewhere (3).  For all experiments, nuclei were digested with 40 and 160
units of Cla I, and equal cutting of each nuclei preparation by the two different amounts
of Cla I was observed, indicating complete digestion of the accessible templates.
Digestion of nuclei with micrococcal nuclease (MNase) revealed that nucleosomes are
appropriately positioned under repressing conditions at the PHO5 promoter in the arg82-
153 mutant (2).
MS# 1078062, Supplementary Online Material 5
Figure S2.  IP4, IP5, and IP6 levels are greatly reduced in the arg82-153 mutant,
suggesting that there is a defect in IP3 kinase activity in this strain.  HPLC analysis of
soluble extracts prepared after steady-state [3H]inositol labeling (4) of pho81∆ pho80ts
(wild-type) and arg82-153 pho81∆ pho80ts strains, grown in synthetic high phosphate
medium at the permissive temperature (25°C).  The elution positions of IP3, IP4, IP5, and
IP6 are indicated.  The predominant IP isomer in wild-type yeast from steady-state
[3H]inositol labeling is IP6, whereas cells lacking Arg82 show a loss of IP4, IP5, and IP6,
and have increased amounts of IP2 and IP3 (5).
Figure S2
0
500
1000
1500
12 24 36 48 60 72
Time (min)
Wild type
arg82-153
IP6
IP3
IP2
IP5IP4
MS# 1078062, Supplementary Online Material 6
Figure S3.  ChIP was performed as described elsewhere (6).  Strains were grown under
repressing conditions (DMSO) or shifted to inducing conditions by adding 10 µM 1-
Figure S3
A
C
B
Intensity
-dI/dT
MS# 1078062, Supplementary Online Material 7
NaPP1 for 30 min.  Ino80-HA and Snf2-Myc were tagged at the C-terminus with 3 HA
and 13 Myc epitopes, respectively, and the gene coding for each was integrated at the
native locus.  The PHO5 (-492 to -191 base pairs (bp)), PHO84 (-460 to -213 bp), and
ACT1 (-142 to +35 bp) promoter DNA sequences were amplified by PCR and quantified
by a DNA Engine Opticon System (MJ Research) for continuous fluorescence detection.
Each primer pair produced a single DNA fragment of the appropriate size and no
detectable primer dimers.  (A) Data graph.  Input DNA from a randomly-selected ChIP
sample was diluted 100-, 500-, 2500-, and 12,500-fold, and an equal volume from each
dilution was added to samples containing the PHO5, PHO84 or ACT1 primers and
subjected to PCR.  The fluorescence of SYBR Green I, which is greatly enhanced upon
binding dsDNA, is graphed versus cycle number for all 12 reactions.  For a given
dilution, the PHO5, PHO84 and ACT1 primers yield curves that are nearly
superimposable, indicating that each amplifies its target with similar efficiency.  The
position of the cycle threshold (C(T)) is indicated by the dashed line.  (B) Standard curve.
The initial quantities of template in the serial dilutions were arbitrarily set to 100, 20, 4,
and 0.8 to graph the Log Quantity versus the cycle at which the sample’s fluorescence
crosses the C(T) line.  The R-square value of 0.998 indicates that the fit of the linear
curve explains 99.8% of the variation in the data.  Data from all 12 reactions (i.e. from
each primer pair) were used to generate the curve.  For the experiments in the manuscript,
PCR samples of a particular primer pair were quantified using standard curves generated
from the same primer pair.  (C) Melting curve.  Following PCR, the fluorescence
intensity (I) was graphed versus temperature (T) for a PHO5 (green), PHO84 (red) and
ACT1 (blue) sample.  A decrease in fluorescence is observed as the dsDNA melts.  Each
MS# 1078062, Supplementary Online Material 8
of the PHO5, PHO84 and ACT1 PCR fragments produces a distinct melting curve
corresponding to fragment length and GC content.  A single peak generated by graphing
–dI/dT versus temperature for each indicates product homogeneity.
Figure S4
A
0
0.5
1
1.5
2
2.5
3
3.5
WT pho4 delete arg82 delete
DMSO
1-NaPP1
B
0
5
10
15
20
25
30
35
40
45
50
WT pho4 delete arg82 delete
DMSO
1-NaPP1
MS# 1078062, Supplementary Online Material 9
Figure S4.  (A) ChIP using a monoclonal antibody that recognizes Ino80-HA in
Pho85F82G-expressing strains with the relevant genotypes indicated.  (B) ChIP using
affinity-purified polyclonal antibodies recognizing Pho4 in Pho85F82G-expressing strains
with the relevant genotypes indicated.
Figure S5.  PHO5 promoter activity is decreased by mutations in UASp1 or UASp2.
Northern analysis of Pho85F82G-expressing strains carrying base substitutions in the
Pho4-binding sites of UASp1 and UASp2 in the PHO5 promoter.  Mutation of UASp1
resulted in a promoter that is uninducible, while mutation of UASp2 decreased promoter
activity 2.5-fold.  AAGCTT was substituted for either CACGTT (UASp1) or CACGTG
(UASp2), and the mutant alleles were integrated at the native locus.  Strains were grown
Figure S5
PHO5
ACT1
PHO84
1-NaPP1 + + +
WT
UASp1
mutant
UASp2
mutant
MS# 1078062, Supplementary Online Material 10
in repressing conditions (-) or shifted to inducing conditions (+) by adding 10 µM 1-
NaPP1 for one hour.
Figure S6
MS# 1078062, Supplementary Online Material 11
Figure S6.  Schematic model illustrating the activation of PHO5 transcription by ATP-
dependent chromatin remodeling complexes.  Following import into the nucleus, Pho4
and Pho2 bind to UASp1 in the nucleosome-free region of the unremodeled promoter.  In
the presence of IP4/IP5, Pho4 and/or Pho2 recruit(s) the INO80 complex (and possibly
SWI/SNF) to the promoter.  This leads to disruption of positioned nucleosomes –1 to –4,
greatly increasing the accessibility of UASp2 and the TATA box for binding by Pho4 and
TFIID, respectively.
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Regulation of Chromatin Remodeling by Inositol Polyphosphates

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Regulation of Chromatin Remodeling by Inositol Polyphosphates

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