The dual control of TFIIB recruitment by NC2 is gene specific.

Abstract

Negative co-factor 2 (NC2) is a conserved eukaryotic complex composed of two subunits, NC2alpha (Drap1) and NC2beta (Dr1) that associate through a histone-fold motif. In this work, we generated mutants of NC2, characterized target genes for these mutants and studied the assembly of NC2 and general transcription factors on target promoters. We determined that the two NC2 subunits mostly function together to be recruited to DNA and regulate gene expression. We found that NC2 strongly controls promoter association of TFIIB, both negatively and positively. We could attribute the gene-specific repressor effect of NC2 on TFIIB to the C-terminal domain of NC2beta, and define that it requires ORF sequences of the target gene. In contrast, the positive function of NC2 on TFIIB targets is more general and requires adequate levels of the NC2 histone-fold heterodimer on promoters. Finally, we determined that NC2 becomes limiting for TATA-binding protein (TBP) association with a heat inducible promoter under heat stress. This study demonstrates an important positive role of NC2 for formation of the pre-initiation complex on promoters, under normal conditions through control of TFIIB, or upon activation by stress via control of TBP.

Full text

Page 1

Published online 29 November 2007Nucleic Acids Research, 2008, Vol. 36, No. 2539–549
doi:10.1093/nar/gkm1078
The dual control of TFIIB recruitment by NC2 is
gene specific
Patrick Masson1, Elisa Leimgruber1,2, Sandrine Creton1and Martine A. Collart1,*
1Department of Microbiology and Molecular Medicine, CMU, 1 rue Michel Servet 1211, Geneva 4, Switzerland and
2Department of Pathology and Immunology
Received October 19, 2007; Revised November 14, 2007; Accepted November 15, 2007
ABSTRACT
Negative co-factor 2 (NC2) is a conserved eukaryotic
complex composed of two subunits, NC2a (Drap1)
and NC2b (Dr1) that associate through a histone-fold
motif. In this work, we generated mutants of NC2,
characterized target genes for these mutants and
studied the assembly of NC2 and general transcrip-
tion factors on target promoters. We determined
that the two NC2 subunits mostly function together
to be recruited to DNA and regulate gene expression.
We found that NC2 strongly controls promoter
association of TFIIB, both negatively and positively.
We could attribute the gene-specific repressor
effect of NC2 on TFIIB to the C-terminal domain of
NC2b, and define that it requires ORF sequences
of the target gene. In contrast, the positive function
of NC2 on TFIIB targets is more general and requires
adequate levels of the NC2 histone-fold heterodimer
on promoters. Finally, we determined that NC2
becomes limiting for TATA-binding protein (TBP)
association with a heat inducible promoter under
heat stress. This study demonstrates an important
positive role of NC2 for formation of the pre-initiation
complex on promoters, under normal conditions
through control of TFIIB, or upon activation by stress
via control of TBP.
INTRODUCTION
Transcription by RNA polymerase II is critically depen-
dent upon general transcription factors (GTFs) that allow
the specific association of the polymerase with promoter
regions. Amongst these, the TATA-binding protein (TBP)
binds to promoters and plays a critical role in the
nucleation of the pre-initiation complex (PIC) (1). It
allows the recruitment of both TFIIA and TFIIB,
followed by the other GTFs. Several factors that control
transcription initiation, interact with TBP and either
modify the association of TBP with DNA, or prevent
the association of subsequent GTFs. One such factor is
negative cofactor 2 (NC2), bearing two subunits (NC2a or
Drap1 and NC2b or Dr1), which forms a stable complex
with TBP on promoters (2). Biochemical and genetic data
have suggested that the association of NC2 with DNA-
bound TBP competes with the association of TFIIA and
TFIIB, and thus inhibits transcription initiation (3–8).
NC2 is conserved in eukaryotes, and a crystal structure of
a human NC2–TBP–DNA complex has been resolved (8).
NC2 dimerizes through histone-fold domains (HFD) of
the H2A/H2B type, and the NC2 histone-fold is localized
underneath the DNA surface to which TBP binds. Origi-
nally it was suggested that the carboxy terminal extension
of NC2b might sterically hinder the association of TFIIB
with TBP, whilst regions of NC2a missing in the structure,
C-terminal to the HFD, might be responsible for sterically
affecting the association of TFIIA with TBP. In contrast
to the mutually exclusive binding to TBP proposed for
both TFIIA and NC2, a more recent superposition of
structures has suggested that TFIIA and NC2 could be
bound to TBP simultaneously, albeit with lower affinity
than for either molecule alone (9).
In addition to this simple and quite well-defined model
for transcriptional repression by NC2, many studies have
revealed that NC2 function is complex. Indeed, NC2 has
been demonstrated not only to repress, but also to activate
transcription, in vitro and in vivo (6,10–13). The mechanism
by which NC2 promotes transcription has not been studied
much, and remains unclear. The C-terminal domain of
Drosophila NC2b, essential for repression by NC2, is not
required for activation by NC2 (11). This observation
would suggest the existence of different functional domains
within NC2. A different study has suggested that different
functional forms of NC2 might exist. Indeed, purification
of NC2a and NC2b subunits from Saccharomyces
cerevisiae revealed that the two NC2 subunits could not
be co-purified from yeast cells growing exponentially,
whilst they could be co-purified after glucose depletion
(14). Furthermore, relative cross-linking of the NC2
subunits to a same promoter was different before and
Present address:
Sandrine Creton, Max Planck Institute of Biochemistry, Department of Molecular Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany
*To whom correspondence should be addressed. Tel: 004122 3795476; Fax: 004122 3795702; Email: martine.collart@medecine.unige.ch
? 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Page 2

after glucose depletion suggesting that different forms of
NC2 complexes might be able to associate with promoter
DNA. What these different forms are remains unknown. In
human, evidence has been provided to suggest that NC2a
andNC2b can associate with different proteins, since it was
demonstrated that BTAF1, the human homolog of yeast
Mot1p, interacts with NC2a but not NC2b or the NC2
heterodimer (15). With regard to the mechanism by which
NC2 might associate with DNA, it has been documented
that NC2 associates with DNA-bound TBP, and this has
been studied in vitro using TATA-containing DNA, but
efficient binding of NC2–TBP to DNA that lacks a
canonical TATA box has also recently been demonstrated
(16). This observation may be related to former experi-
ments showing that in yeast, NC2 was required for
transcription from the TATA-less promoter of HIS3 but
repressed transcription from the HIS3 TATA promoter
(13). Finally, while the activity of NC2 has generally been
thought to be related to TBP function, it was recently
demonstrated in vitro that recombinant human NC2, like
other histone-fold complexes, could facilitate nucleosome
assembly by ACF, independently of a direct interaction
with ACF (17). Furthermore, Drosophila NC2b has been
described as a partner of the HFD protein TAF11 in two
hybrid experiments (18).
Inthiswork,usingmutantsofNC2,wehavebeenableto
define NC2 target genes, and determine how NC2 controls
the assembly of the PIC on target genes in vivo. We
demonstrate that in cells growing exponentially, NC2
regulates theassociation of TFIIBto promoters in different
ways depending upon the gene. Furthermore, our study
defines a role for NC2 in the stable recruitment of TBP to
heat inducible genes upon heat stress. Taken together, our
results reveal an important role for the NC2 heterodimer in
the efficient assembly of GTFs to promoters.
MATERIALS AND METHODS
Strainsand growth media
The strains used in this study are listed in Table 1. All
media were standard, and YPD was used for glucose-rich
medium. HSP12and RPS14b were disruptedby
transformation of a PCR-amplified marker TRP1 cassette
according to Longtine (19).
Microarrays
Wild-type (wt) and mutant cells were grown exponentially
at 308C in YPD (2% glucose) during 24h until an OD600
of ?0.8. Cells were harvested and total RNA extracted
by the acid phenol method. The quality of total cellular
RNA was tested on a RNA 6000 Nano Chip (Agilent).
Total cellular RNA was next purified with RNeasy Mini
Handbook kit (QIAGEN). Then, the synthesis of cDNA,
cRNA, the hybridization and the scan of the chip were
performed according to the technical manual of Affimetrix
(GeneChip Expression Analysis). Briefly, oligo dT was
added to 15mg of purified total RNA. After 10min. of
incubation at 708C in first strand buffer with DTT
(10mM), dNTP (500mM each) and reverse transcriptase
(SuperScript II from Invitrogen at 200U/ml) were added
for the first strand cDNA synthesis (1h at 428C). For the
second strand synthesis, dNTP (200mM each), Escherichia
coli DNA ligase (10U), E. coli DNA polymerase I (40U)
and E. coli RNaseH (2U) were added to the second strand
buffer and incubated for 2h at 168C. Finally, T4 DNA
polymerase (10U) was added to the reaction. The cDNA
was purified and after ethanol precipitation, 15mg of
purified cDNA were incubated during 5h at 378C with
HY buffer (Enzo), biotine-coupled ribonucleotides, DTT,
an RNase inhibitor mix and T7 RNA polymerase. The
cRNA was then purified in the same way as total RNA
and 20mg of cRNA were fragmented in 5? hybridization
buffer (200mM Tris–acetate pH 8.1, 500mM KOAc,
150mM MgOAc) and incubated during 35min at 958C.
Hybridization of cRNA. Fifteen micrograms of cRNA
were hybridized to the Yeast Genome S98 chip, during
16h at 458C with 1? hybridization buffer (100mM MES;
1M [Na+]; 20mM EDTA; 0.01 Tween-20), oligonucleo-
tide B2 (50pM), eukaryotic hybridization controls (20?),
herring sperm DNA (0.1mg/ml) and acetylated BSA
(0.5mg/ml). The chip was washed in the Fluidics station,
first with buffer A (6? SSPE, 0.01% Tween-20) then with
buffer B (100mM MES, 0.1M [Na+], 0.01% Tween-20).
Table 1. Strain list
Strain nameGenotypeReference
MY1
MY3298
MY3357
MY3717
MY3718
MY3767
MY3765
MY3721
MY3802
MY4401
MY4312
MY5130
MY5661
MY5662
MY5709
MY5710
MATa gcn4D ura3-52 trp1D1 leu2::PET56 gal2
Isogenic to MY1 except MAT? his3::TRP1 bur6-5
Isogenic to MY1 except MAT? ncb2-2
Isogenic to MY1 except bur6-5 tfa1::TFA1-MYC10-KanMX4
Isogenic to MY1 except tfa1::TFA1-MYC10-KanMX4
Isogenic to MY1 except MAT? ncb2-2 tfa1::TFA1-MYC10-KanMX4
Isogenic to MY1 except bur6-5 tfb3::TFB3-MYC10-KanMX4
Isogenic to MY1 except tfb3::TFB3-MYC10-KanMX4
Isogenic to MY1 except MAT? ncb2-2 tfb3::TFB3-MYC10-KanMX4
Isogenic to MY3298 except sua7::SUA7-MYC10-KanMX4
Isogenic to MY1 except sua7::SUA7-MYC10-KanMX4
Isogenic to MY1 except MAT? ncb2-2 sua7::SUA7-MYC10-KanMX4
Isogenic to MY1 except hsp12::TRP1 sua7::SUA7-MYC10-KanMX4
Isogenic to MY5661 except ncb2-2
Isogenic to MY1 except rps14b::TRP1 sua7::SUA7-MYC10-KanMX4
Isogenic to MY5709 except ncb2-2
(14)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
540
Nucleic Acids Research, 2008, Vol. 36, No. 2

Page 3

Antibodies were added next (Normal Goat IgG 0.1mg/ml;
biotinylated antibodies 0.5mg/ml; acetylated BSA 2mg/
ml; 100mM MES; 1M [Na+]; 0.05% Tween-20) followed
by the SAPE solution (Streptavidine–Phycoerythrine
10mg/ml; acetylated BSA 2mg/ml; 100mM MES; 1M
[Na+]; 0.05% Tween-20). Finally, the chip was scanned
and the scan was analyzed by Affymetrix software
(MicroArraySuite, MicroDB, and DataMiningTool) and
Iobion software (Array Assist). The stringent analysis
consisted of using (p0.05 DE1.0) to obtain significant
target genes for each mutant compared to the wt by all
three proposed methods of analysis (RMA, GCRMA
and CHP). Only genes defined as genes significantly
de-regulated in the mutant relative to the wt by all three
analyses were considered. For comparison, a less-stringent
analysis consisted of using the Affymetrix software,
and looking for genes de-regulated in 100% of the
comparisons (each mutant duplicate compared to each
wt duplicate), without introducing an additional minimal
fold increase or decrease. Once target genes for each
mutant were defined by the Array Assist software,
for each target gene we determined whether in each
duplicate for each mutant it was expressed at higher levels
than for either duplicate of the wt (Supplementary
Table 1).
Chromatine immunoprecipitation (ChIP)
ChIP experiments were performed as described in (14).
Briefly, wt and mutant cells were grown exponentially at
308C in YPD (2% glucose) during 24h until an OD600
of ?0.8. The cells were fixed with 1% formaldehyde
during 20min. at RT, and glycine was added to a final
concentration of 125mM to stop the reaction. The cells
were washed twice (20mM Tris–HCl pH 7.5; 200mM
NaCl) and broken in lysis buffer (50mM HEPES–KOH
pH 7.5; 140mM NaCl; 1mM EDTA pH 8; 1% Triton;
0.1% sodium deoxycholate; 1mM PMSF) during 30min
and sonicated. The size of fragmented chromatin was
verified on a gel to be between 200 and 400bp, then a
fraction of the extracts (input) was incubated over night at
48C with specific antibodies and protein G-Sepharose. The
Sepharose beads were collected and washed with TSE 150
(20mM Tris–HCl pH 8; 2mM EDTA pH 8; 1% Triton;
0.1% sodium deoxycholate; 150mM NaCl), followed by
TSE 500 (20mM Tris–HCl pH 8; 2mM EDTA pH 8; 1%
Triton; 0.1% sodium deoxycholate; 300mM NaCl), then
Buffer III (10mM Tris–HCl pH 8; 1mM EDTA pH 8;
250mM LiCl; 1% Igepal, 0.1% sodium deoxycholate) and
finally 2?TE. Immunoprecipitated chromatin was eluted
in 1% SDS; 50mM Tris–HCl pH 7.5; 10mM EDTA pH
8. Then 10mg/ml of proteinase K was added to the
precipitates and to a fraction of the input during 5h at
658C to reverse cross-links. DNA was extracted and
precipitated. Specific promoter DNA in the input and
precipitates was measured by real-time PCR using SYBR
Green (Eurogentec). The sequences of specific oligonuc-
leotides are available upon request. 9E10 monoclonal
anti-MYC antibodies were purchased from Covance,
and mouse monoclonal anti-Rpb3p and anti-Rpb4p
antibodies from Neoclone. The ChIP experiments were
performed three times and the results are the fold (increase
or decrease) change of the mutant versus the average
wt value.
Co-immunoprecipitation experiments
Cultures were grown exponentially to an OD600of 0.8.
The equivalent of 50 OD600of cells were harvested by
centrifugation for 1min at 13000r.p.m. After washing in
water, cell pellets were frozen at ?808C, thawed and
resuspended in 600ml of buffer B (40mM HEPES–KOH
pH 7.5, 150mM KOAc, 100mM KCl, 20% glycerol,
1? protease inhibitor cocktail and 1mM PMSF).
Cells were broken by the addition of 300ml of glass
beads and vortexing vigorously for 15min. Whole-cell
extracts (WCE) were clarified by centrifugation for
15min. at 13 000 r.p.m. and at 48C. The protein
concentration was determined by the Bradford assay.
For co-immunoprecipitation experiments, 1ml of anti-
NC2a antibody was mixed with 1mg of cell extract and
20ml of protein G-Sepharose beads over night at 48C in a
total volume of 250ml. The beads were washed three times
with 40mM HEPES–KOH pH 7.5, 150mM KOAc,
100mM KCl, 20% glycerol before elution with boiling
in loading buffer.
S1analysis
Total cellular RNA extraction and S1 analyses were
performed as described previously (14). The sequences of
the oligonucleotides used are available upon request.
RESULTS
C-terminal extensions following theHFDs of NC2aand
NC2bare importantfor cell growth
Previous studies have suggested that the NC2 a- and
b-subunits might have different roles in cells growing
exponentially, and that the function of NC2 is regulated
by glucose (14). Indeed, in yeast, differences in associa-
tion of NC2 subunits with DNA, and changes in
co-purification of the NC2 subunits, were observed
between growth in high or low glucose. To further
characterize the transcriptional functions of the two
subunits of NC2, we generated two mutant strains. The
first strain expresses from its endogenous locus and
promoter, a NC2b mutant truncated at the C-terminus
after the HFD and fused to a triple HA epitope (MY3357,
bearing the ncb2-2 allele and expressing the NC2b?122
protein, Table 1). The second strain expresses from its
endogenous locus and promoter a mutant NC2a trun-
cated at the C-terminus after the HFD and fused to a
triple HA epitope (MY3298, bearing the bur6-5 allele and
expressing the NC2a?120protein, Table 1). Both mutant
strains were cold and temperature sensitive for growth,
and the ncb2-2 mutant additionally grew slowly even at
308C in rich medium (Figure 1A). We next analyzed
the profile of transcripts genome-wide in wt, bur6-5 and
ncb2-2 cells that we kept growing exponentially for 24h in
rich medium and high glucose (2%). We performed a
stringent analysis that considered only genes whose
Nucleic Acids Research,2008, Vol. 36,No. 2 541

Page 4

expression was significantly different in the mutant
compared to the wt (Array Assist software, Iobion, see
Materials and Methods section). This stringent analysis
defined 133 target genes for the C-terminal deletion
mutant of NC2a (bur6-5) and 169 target genes for the
C-terminal deletion mutant of NC2b (ncb2-2) (Figure 1B
and Supplementary Table 1). Genes were both up- and
downregulated in the bur6-5 and ncb2-2 mutants, and
there was a significant overlap in the genes de-regulated in
both mutants (Figure 1B). These results support a strong
interplay between both subunits since, in addition to the
important overlap of target genes between the two
mutants, many genes that were affected in the ncb2-2
mutant only, are actually genes that respond to a change
in growth rate such as ADE12, ADE13 and ADE17, and
only the ncb2-2 mutant grew slowly under the conditions
of analysis (see Figure 1A growth of ncb2-2 versus wt
and bur6-5).
Independent mRNA analysis confirms majoritytarget
genesidentified by microarrays
To obtain confirmation of the results found by the
microarray analyses, we analyzed by S1 digestion the
mRNA levels of some of the target genes defined. In
support of the microarray experiments, BUR6, the gene
coding for NC2a itself, was upregulated in both mutants
compared to the wt (Figure 2A). For the general stress
genes HSP12 and HSP26, the microarray data showed an
induction in both mutants, whereas S1 analysis suggested
a strong increase of HSP26 mRNA in both mutants but
only in the ncb2-2 mutant for HSP12 (Figure 2B and C).
MUC1 (also termed FLO11), encoding a cell surface
glycoprotein required for diploid pseudohyphal formation
and haploid invasive growth, is the gene whose mRNA
was the most induced in the ncb2-2 mutant according to
the microarray experiments (around 77-fold increase).
We could confirm a strong upregulation or this mRNA by
S1 analysis (Figure 2D), and similarly we were able to
confirm the upregulation of DAN1 in the ncb2-2 mutant
(Figure 2E). We were also able to confirm decreased
expression of several genes in the mutants compared to the
wt: RPS14b (Figure 2F), RPS28b, RPS9a and BNA4
(data not shown) as well as genes whose expression was
not affected in any of the mutants such as ADH1 or PHO4
(Figure 2G and H).
Global loss ofNC2 on promoters inthe two mutants
The next step in our analysis of the mutants was to define
what was happening with the NC2 subunits on the
promoters of the target genes defined earlier. We thus
compared the association of NC2a and NC2b to DNA in
wt and mutant cells by ChIP experiments. We found that
there was a significant reduction in cross-linking of both
NC2 subunits to DNA, whether we checked genes
upregulated in the mutants such as HSP12 and BUR6
(Figure 3A), genes downregulated in the mutants such as
RPS14b (Figure 3A) or genes unaffected in the mutants
(data not shown).
Since differences in cross-linking of both NC2 subunits
to DNA in both mutant strains were observed, we next
analyzed the expression and interaction of the NC2
subunits in wt cells and in cells expressing the mutant
forms of the NC2 subunits (Figure 3B). In bur6-5, wt
levels of NC2a could be immunoprecipitated with NC2a
antibodies (lane 5, upper panel), despite lower levels of
the protein in the total extract of mutant cells compared to
wt cells (compare lanes 1 and 2, upper panel), but no
detectable NC2b was co-immunoprecipitated (lane 5,
lower panel), although NC2b was expressed at wt levels
in this strain (compare lanes 1 and 2, lower panel).
Furthermore, high levels of NC2b could be immunopre-
cipitated with NC2b antibodies in this strain (lane 8, lower
panel), but only very low levels of NC2a co-immuno-
precipitated (lane 8, upper panel). In ncb2-2, NC2b is
expressed at only very low levels (lane 3, lower panel), and
thus whether NC2a or NC2b is immunoprecipitated (lane
6, upper panel; or lane 9, lower panel), only very low levels
of the other subunit is detectable in the immunoprecipitate
(lane 9, upper panel and lane 6, lower panel). These results
demonstrate that in both mutant strains, there are only
low levels of the NC2 heterodimer, which correlates nicely
with reduced cross-linking of both subunits to DNA
in both strains, in contrast to wt expression of NC2b in
bur6-5 and NC2a in ncb2-2.
The general changes in transcription forthemutants
In order to analyze the status of the PIC on target
promoters in the strains expressing the mutant forms of
NC2, we investigated the presence of different factors of
the transcription machinery using ChIP experiments. The
first protein of the PIC we chose to look at was the TBP.
Indeed, NC2 has previously been found to accelerate TBP
binding to promoters and stabilize TBP–DNA complexes
(16). Interestingly, we found that on HSP12 no significant
Figure 1. Analysis of bur6-5 and ncb2-2 growth and definition of target
genes by microarray experiments. (A) The indicated strains were
streakedonglucose-richmedium
temperatures for 3–7 days. (B) Venn diagrams indicating the number
of genes that were defined as induced or repressed in bur6-5 or ncb2-2
using the Array Assist software. The overlap between the two mutants
was defined by determining for each of the target genes of one mutant
whether each duplicate of the other mutant was different from each
duplicate of the wild-type, with an average of 2-fold difference
(data provided in Supplementary Table 1).
and placedattheindicated
542
Nucleic Acids Research, 2008, Vol. 36, No. 2

Page 5

Figure 3. Association of NC2 subunits with DNA in the bur6-5 and ncb2-2 mutants. (A) Wild-type or mutant cells as indicated were grown in high
glucose for 24h, cross-linked and cell extracts were incubated with antibodies against NC2a or NC2b. After immunoprecipitation and purification
of nucleic acids in the immunoprecipitates, the amount of the indicated promoters (HSP12, BUR6 or RPS14b) in the precipitates was evaluated by
real-time PCR, and referred to the input extract. The experiments were performed three times independently and the results are presented as the fold-
change in the mutant over the average wild-type. (B) Exponentially growing cells from the strains indicated above the panels were lyzed for total
protein extract (TE) preparation. The extracts were incubated with antibodies against NC2a (IP NC2a) or N2b (IP NC2b) followed by protein G
Sepharose, and the total extract or immunoprecipitates were analyzed by western blot with polyclonal NC2a (upper panel) or NC2b (lower panel)
antibodies as indicated. All samples tested with one antibody were run on the same gel and exposed together for the same length of time. The
position of the proteins encoded by bur6-5 and ncb2-2, as well as the proteins encoded by the wild-type genes are indicated on the right of the panels,
and on the left, asterisk indicates a band cross-reacting with NC2a antibodies and which co-migrates with one form of NC2a.
Figure 2. mRNA analysis to confirm the target genes found by the microarrays. Cells were grown up to an O.D.600of 0.8, and 50mg of total cellular
RNA was analyzed by S1 digestion for the levels of the indicated transcripts: (A) BUR6, (B) HSP12, (C) HSP26 (D) MUC1, (E) DAN1, (F) RPS14b
(G) ADH1 and (H) PHO4. DED1 were measured in each reaction as an internal control, since the microarray experiments indicated that it was not
affected by the bur6-5 or ncb2-2 mutations.
Nucleic Acids Research,2008, Vol. 36,No. 2543