MOLECULAR AND CELLULAR BIOLOGY, Jan. 2008, p. 30–39
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 1
Nicotinamide Uncouples Hormone-Dependent Chromatin Remodeling
from Transcription Complex Assembly?
Sayura Aoyagi and Trevor K. Archer*
Chromatin and Gene Expression Section, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences,
National Institutes of Health, 111 Alexander Drive, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Received 28 June 2007/Returned for modification 3 August 2007/Accepted 8 October 2007
Sirtuins, homologs of the yeast SIR2 family, are protein deacetylases that require nicotinamide adenosine
dinucleotide as cofactor. To determine whether the sirtuin family of deacetylases is involved in progesterone
receptor (PR)-mediated transcription, the effect of sirtuin inhibitor, nicotinamide (NAM), was monitored in
T47D breast cancer cells. NAM suppressed hormone-dependent activation of PR-regulated genes in a dose-
dependent manner. Surprisingly, NAM-mediated inhibition of PR-mediated transcription occurs indepen-
dently of SIRT1 and PARP1. Chromatin immunoprecipitation experiments did not show that PR binding nor
that of the coactivators CBP and SRC3 was compromised. Consistent with the recruitment of the BRG1
chromatin remodeling complex, promoter chromatin remodeling still occurs despite NAM inhibition of PR
transactivation. Rather, we show that this inhibition of transcription is due to dramatic loss of recruitment of
the basal transcriptional machinery to the promoter. These results show that NAM uncouples promoter
chromatin remodeling from transcription preinitiation complex assembly and suggest the existence of vital
NAM-regulated steps required for promoter chromatin remodeling and basal transcription complex
Steroid hormone receptors such as the progesterone recep-
tor (PR) and the glucocorticoid receptor (GR) are part of a
large nuclear receptor (NR) family of eukaryotic transcription
factors (41, 59). NRs play essential roles in numerous biolog-
ical processes such as growth and development, reproduction,
homeostasis, and metabolism by eliciting a transcriptional out-
put from target genes in response to their cognate ligands
which include steroids, retinoids, thyroid hormone, and vita-
min D3, among others (12, 41, 59). Studies of NR action have
not only provided insight into their physiological roles but have
been vital in the overall understanding of the mechanism of
transcription by transcription activators (34, 42, 49, 55).
The process of ligand-dependent transcription initiation by
steroid hormone receptors (SHRs) such as PR and GR involve
ligand binding, followed by receptor binding to the hormone
responsive elements at the promoter DNA of target genes as
dimers. The promoter-bound SHR leads to recruitment of a
large number of coactivators that work in sequence and/or in
combination to ultimately facilitate the recruitment of RNA
polymerase II (RNAP II) and the transcription machinery to
elicit a transcriptional response (34, 49, 59). These coactivators
include the p160/SRC family of proteins (SRC1, -2, and -3)
that directly interact with NRs through consensus LXXLL
motifs (NR boxes) (25, 49). The p160/SRC family of proteins
is also able to associate with histone-modifying enzymes such
as histone acetyltransferase p300/CBP and histone methyl-
transferase CARM1, thereby playing a role in bridging the
recruitment of chromatin modifiers to SHR target promoters
(34, 49, 56). In addition to the p160/SRC family of proteins, PR
and GR both also interact with and recruit the ATP-dependent
chromatin remodeling complex, SWI/SNF to target promoters
to render the promoter chromatin more accessible, allowing
additional transcription factors, coactivators, and the general
transcription factors access the promoter DNA (2, 6, 21, 34).
The SHRs also recruit the multisubunit Mediator complex
(also known as TRAP/DRIP/ARC/CRSP/SMCC complexes)
(5). The Mediator complex has been purified by various bio-
chemical methods and have been found to interact directly
with various NRs, including thyroid hormone receptor (19),
vitamin D receptor (51, 52), estrogen receptor (ER) (30), and
androgen receptor (AR) (63), as well as GR (26). These con-
tacts are made through the NR box motif (LXXLL) containing
subunit MED1 (also known as TRAP220, ARC/DRIP205,
PBP, and CRSP200 but referred to here by the MED acronym
nomenclature set forth by Bourbon et al. ), as well as
MED14 (also known as TRAP170, ARC/DRIP150, and p110)
in the case of GR (19, 26, 66). The Mediator complex is
thought to aid in the recruitment RNAPII and the formation
of the transcription preinitiation complex (PIC) machinery to
ligand-activated promoters. Many of the RNAPII regulated
genes appear to require the Mediator complex for gene ex-
pression, with specific subunits of the complex playing distinct
roles in regulating target genes through their interactions with
various transcription activators and the RNAPII transcription
machinery (15, 35, 40).
Adding to the complexity of the combinatorial recruitment
of large numbers of coactivators for gene activation is the
regulation of transcription factor and coactivator activities
themselves by posttranslational modifications. Some examples
include SHR, which undergoes several types of modifications
such as acetylation, phosphorylation, ubiquitylation, and
* Corresponding author. Mailing address: Chromatin and Gene
Expression Section, Laboratory of Molecular Carcinogenesis, NIEHS/
NIH, 111 Alexander Drive, P.O. Box 12233 (MD D4-01), Research
Triangle Park, NC 27709. Phone: (919) 316-4565. Fax: (919) 316-4566.
?Published ahead of print on 22 October 2007.
sumoylation that affect receptor activity and stability (18). p300
acetylates promoter chromatin, as well as autoacetylates itself,
which leads to p300 dissociation and enhancement of TFIID
binding (8). In addition, NR coactivator ACTR (SRC3) is
acetylated by p300/CBP, which leads to disruption of the
ACTR (SRC3) interaction with ER and coincides with cessa-
tion of transcription (13).
In the present study, we were interested in identifying novel
factors that may influence PR-mediated transcription. In par-
ticular, we focused on sirtuins, homologs of the yeast SIR2
family, which are protein deacetylases that require nicotin-
amide adenosine dinucleotide (NAD?) as a cosubstrate (9, 16,
27). The closest mammalian structural ortholog of the yeast
Sir2 protein, SIRT1, exerts its effects on a wide range of cel-
lular metabolism by acting as a deacetylase of histones, as well
as nonhistone substrates such as PCAF, p300, p53, PGC-1?,
AR, and possibly ER? (22, 32, 65). The effect of the sirtuin
inhibitor, nicotinamide (NAM) (4, 17), on PR-mediated tran-
scription was monitored in T47D human breast cancer cells to
determine whether the sirtuin family of deacetylase is involved
in PR-mediated transcription. While NAM is a known sirtuin
inhibitor, it is also known to inhibit the poly-ADP-ribosylation
(PAR) activities of poly-ADP-ribose polymerase (PARP) fam-
ily of proteins (37). PARP1, the best characterized member of
the PARP family, has been closely linked to transcription
through various mechanisms such as influencing chromatin
structure, ribosylation of transcription factors, and altering
Mediator activities, in addition to its role in DNA repair (29,
31, 33, 48). Our data show that NAM suppresses hormone-
dependent activation of PR-regulated genes in a dose-depen-
dent manner. However, unexpectedly, small interfering RNA
(siRNA) knockdown experiments demonstrate that NAM in-
hibition of PR mediated transcription occurs independently of
SIRT1 and PARP1. The inhibition of PR-mediated transcrip-
tion is due to a dramatic loss of recruitment of the basal
transcriptional machinery (RNAPII, TBP, Mediator) as deter-
mined by chromatin immunoprecipitation (ChIP) assays, while
hormone-dependent association of PR and various coactiva-
tors at the promoter is not compromised in the presence of
NAM. Interestingly, restriction enzyme hypersensitivity assay
shows that promoter chromatin remodeling still occurs despite
NAM inhibition of PR transactivation. These results suggest
that NAM inhibits the coordination of basal transcription ma-
chinery assembly after chromatin remodeling of the promoter.
Chromatin remodeling therefore must be followed by distinct
critical steps in which the remodeling event is efficiently com-
municated with the transcription PIC formation event.
MATERIALS AND METHODS
Cell culture and siRNA transfections. T47D/2963.1 (2963.1) cells were derived
from human T47D breast cancer cells by stable transfection of the chimeric
bovine papillomavirus-based construct pJ83d carrying the mouse mammary tu-
mor virus (MMTV) long terminal repeat (LTR) attached to the bacterial chlor-
amphenicol acetyltransferase (CAT) gene (45). T47D/A1-2 (A1-2) cells were
derived from human T47D breast cancer cells by stable transfection of the GR
expression plasmid pGRneo and the plasmid pHHLuc that contains the MMTV-
LTR sequences attached to the luciferase gene (47). Cells were grown at 37°C
with 5% CO2in Dulbecco modified Eagle medium (Invitrogen, Carlsbad, CA)
containing 10% fetal bovine serum (HyClone, Logan, UT) supplemented with 10
mM HEPES and 2 mM glutamine (Invitrogen).
RNA isolation and reverse transcription-PCR (RT-PCR). 2963.1 and A1-2
cells grown in six-well plates were treated with NAM or vehicle control (H2O) for
30 min, followed by treatment with hormone or ethanol (EtOH) for 4 h as
indicated in the figure legends. Total RNA was isolated by using TRIzol reagent
(Invitrogen) according to the manufacturer’s protocol. 2 ?g of total RNA was
used to perform the RT reaction according to the First-Strand synthesis proto-
cols (Invitrogen). PCR analysis was performed by real-time PCR with the fol-
lowing primers: MMTV-Nucleosome A (5?-AGT CCT AAC ATT CAC CTC
TTG TGT GT-3? and 5?-ACC CTC TGG AAA GTG AAG GAT AAG T-3?),
GAPDH (5?-TCG GAG TCA ACG GAT TTG G-3? and 5?-GGC AAC AAT
ATC CAC TTT ACC AGA GT-3?), CEBP/? (5?-CGT GCC CGC TGC AGT
T-?3 and 5?-CTC GCA GTT TAG TGG TGG TAA GTC-3?), SGK (5?-GAC
CCC GAG TTT ACC GAA GAG-3? and 5?-GGA AAG CCT CGG CAG
CTT-3?), and EZF (5?-CGC TCC ATT ACC AAG AGC TCA T-3? and 5?-CGA
TCG TCT TCC CCT CTT TG-3?). Real-time PCRs were performed by using the
Stratagene SYBR green QPCR master mix and Stratagene Mx3000p instrument
(La Jolla, CA). After the gene transcript levels were normalized by that of
GAPDH, the level of transcription in the absence of any treatment (?siRNA,
?NAM, ?hormone) was set to 1 as described in the figures. The data presented
are an average of three independent experiments with the standard mean error
siRNA transfections. 2963.1 and A1-2 cells were seeded in six-well plates
(105/well) and grown overnight. 2963.1 cells were transfected with 100 pmol of
siRNA against nontargeting scrambled sequence (Dharmacon, Lafayette, CO),
SIRT1 (Dharmacon), or PARP1 (Santa Cruz Biotechnology, Santa Cruz, CA) or
50 pmol each of SIRT1 and PARP1 siRNA, while A1-2 cells were transfected
with 200 pmol of nontargeting scrambled sequence siRNA or 100 pmol each of
MED1 and MED14 siRNA (Dharmacon) per well, using Lipofectamine 2000
reagent (Invitrogen) as according to the manufacturer’s protocol for 48 h.
Western blot analysis. 2963.1 and A1-2 cells were lysed in buffer X (100 mM
Tris-Cl [pH 8.5], 250 mM NaCl, 1% [vol/vol] Nonidet P-40, and 1 mM EDTA)
with protease inhibitor cocktail (Sigma, St. Louis, MO). Proteins were electro-
phoresed on 6% or 4 to 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels
and transferred to polyvinylidene difluoride membranes (Invitrogen). Mem-
branes were probed with antibodies to SIRT1 (H-300), PARP1 (F-2), RNAP II
(N-20), TBP (SI-1), and MED1/TRAP220 (M-255) (all from Santa Cruz Bio-
technology), MED14/DRIP150 (gift from M. Garabedian), and ?-tubulin
ChIP assay. 2963.1 cells (1.2 ? 107) were seeded in 15-cm diameter tissue
culture plates and treated the next day with 50 mM NAM or vehicle (H2O) for
30 min, followed by treatment with 10 nM R5020 or vehicle (EtOH) for 1 h. The
cells were then fixed with 1% formaldehyde at 37°C for 10 min. Cells were
collected by centrifugation in phosphate-buffered saline containing protease
inhibitors. Nuclei were isolated as previously described (36), lysed in SDS lysis
buffer, followed by the ChIP assay as described by Upstate Biotechnology. Im-
munoprecipitation was performed overnight with antibodies to PR (H-190),
NF-1 (H-300), BRG1 (H-88), CBP (A-22), SRC3/NCoA-3 (M-397), RNAP II
(N-20), and TBP (SI-1) (all from Santa Cruz Biotechnology) and MED1/
TRAP220 and MED14/DRIP150 (gifts from M. Garabedian). For acetylated
histone ChIP assays, 2963.1 cells (2 ? 106) were seeded in 10-cm-diameter tissue
culture plates and treated the next day with 50 mM NAM or vehicle (H2O) for
15 min, followed by treatment with 10 nM R5020 or vehicle (EtOH) for 30 min.
The cells were then fixed with 1% formaldehyde at 37°C for 10 min. Cells were
collected by centrifugation in PBS containing protease inhibitors and lysed in
SDS lysis buffer. After immunoprecipitation, 60 ?l of salmon sperm DNA-
protein A-agarose was added for 1 h at 4°C to capture the immune complexes.
The agarose beads were washed, chromatin extracted, and protein-DNA cross-
link reversed, and the proteins were digested by proteinase K as indicated in the
Upstate ChIP assay protocol. DNA was purified by QIAquick PCR purification
kit (Qiagen, Valencia, CA) and analyzed by real-time PCR analysis using Stra-
tagene SYBR green QPCR master mix and Stratagene Mx3000p instrument with
the following primers: Nucleosome B (5?-GGT TAC AAA CTG TTC TTA AAA
CGA GGA T-3? and 5?-CAG AGC TCA GAT CAG AAC CTT TG-3?) or
Nucleosome A (sequences listed above). The data presented are the average of
three independent experiments with standard mean of error as indicated.
NAM inhibits PR- and GR-mediated transcription. The sir-
tuins, SIRT1 in particular, have been known to participate in
various endocrine signaling pathways, including those medi-
ated by PPAR?, PGC1?, ER?, and AR (22, 32, 65). We
wanted to examine the possibility of sirtuins being involved in
VOL. 28, 2008NICOTINAMIDE AND PR-MEDIATED TRANSCRIPTION 31
PR-mediated transcription by using an inhibitor of sirtuins,
NAM. NAM is a reaction intermediate of SIRT1 deacetylation
activity that feeds back to inhibit the deacetylation activity of
SIRT1 (4, 17). The effect of NAM on PR-mediated transcrip-
tion was determined by using T47D/2963.1 (2963.1) human
breast cancer cells that endogenously express PR (45) that
were treated with or without NAM for 30 min, followed by
treatment with synthetic progesterone R5020 or vehicle
(EtOH) for 4 h. RT-PCRs were performed from isolated
RNA, and the expression levels of known PR-regulated genes
CEBP/?, EZF and SGK (53, 62) were analyzed by real-time
PCR. The RT-PCR analysis revealed that NAM inhibits hor-
mone-dependent activation of PR-regulated genes in a dose-
dependent manner (Fig. 1A). In addition, to determine
whether this is a phenomenon that is particular to PR activity,
the effect of NAM on the activity of a related receptor GR was
tested in the same T47D human breast cancer cell line back-
ground that expresses rat GR [T47D/A1-2 (A1-2) cells] (47).
RT-PCR analysis of GR- and PR-regulated genes in the A1-2
cell line demonstrated that NAM also inhibits glucocorticoid
dexamethasone (Dex)-induced GR-mediated transcription in
addition to PR-mediated transcription (Fig. 1B).
NAM inhibits PR-mediated transcription independently of
SIRT1 and PARP1. The inhibition of PR-mediated transcrip-
tion by NAM suggested that perhaps SIRT1 and PARP1 are
involved in this event. Both SIRT1 and PARP1 have been
closely linked to NR-mediated transcription (9, 33, 65). To test
this possibility, SIRT1 and PARP1 levels individually or to-
gether were reduced by siRNA treatment in 2963.1 cells, fol-
lowed by analysis of PR-mediated transcription in the presence
FIG. 1. NAM inhibits PR- and GR-mediated transcription. T47D/2963.1 cells (A) and T47D/A1-2 cells (B) were treated with the indicated
amounts of NAM or vehicle (H2O) for 30 min, followed by treatment with R5020 (10 nM), Dex (100 nM), or vehicle (EtOH) for 4 h as indicated.
Total RNA was harvested and analyzed by real-time RT-PCR with primers specific for the indicated genes or GAPDH as a control. The levels
of transcripts for each gene as determined by real-time PCR were normalized to those of GAPDH, and the value for the untreated control (0 mM
NAM, EtOH) was set to 1. The error bars represent the standard error of the mean.
32 AOYAGI AND ARCHERMOL. CELL. BIOL.
or absence of NAM. Efficient reduction of both SIRT1 and
PARP1 protein levels after 48 h of siRNA treatment compared
to treatment with scrambled nontargeted siRNA control were
achieved as demonstrated on the Western blot of prepared
whole-cell extracts in both NAM- and R5020-treated and un-
treated cells (Fig. 2A). After siRNA treatment, the cells were
treated with or without NAM for 30 min, followed by 4 h of
R5020 treatment or vehicle control. The RT-PCR results show
that knockdown of SIRT1 and PARP1 individually or together
leads to a slight decrease in PR-mediated expression of EZF
and SGK. However, these changes are modest and, most im-
portantly, all of the PR-regulated genes analyzed showed a
marked decrease in expression in the presence of NAM in spite
of a significant loss of cellular SIRT1 and PARP1 protein (Fig.
2B and C). This siRNA experiment demonstrates that SIRT1
and PARP1 may play some role in the transcription of a subset
of the genes but that the NAM inhibition of PR-mediated
transcription of the three genes tested occurs independently of
SIRT1 and PARP1.
NAM does not inhibit the PR binding and recruitment of
coactivators to the promoter. Since the siRNA experiment
(Fig. 2) excluded SIRT1 and PARP1 as the targets of NAM-
FIG. 2. NAM inhibition of PR-mediated transcription occurs independently of SIRT1 and PARP1. T47D/2963.1 cells were transfected with
SIRT1, PARP1, both SIRT1 and PARP1 (S&P), or the scrambled nontargeting control (Control) siRNA for 48 h. The cells were then treated with
50 mM NAM or vehicle (H2O) as ?NAM for 30 min, followed by treatment with R5020 (10 nM) or with vehicle (EtOH) as the ?R5020 control
for 4 h. (A) The cellular levels of SIRT1 and PARP1 were determined by Western blot analysis of whole-cell extracts derived from the
siRNA-treated cells. ?-Tubulin was probed as a loading control. (B and C) Total RNA harvested from the siRNA-treated samples was analyzed
by real-time RT-PCR with primers specific for the indicated genes. The levels of transcripts for each of the genes were normalized against that
of GAPDH and the value for the untreated control (scrambled siRNA, ?NAM, ?R5020) set to 1. The “R” represents R5020. The error bars
represent the standard error of the mean.
VOL. 28, 2008 NICOTINAMIDE AND PR-MEDIATED TRANSCRIPTION 33
mediated inhibition of PR-mediated transcription, a series of
ChIP experiments were conducted to determine the mecha-
nism by which NAM inhibits transcription. ChIP experiments
were performed to monitor the recruitment of various cofac-
tors and transcription factors to the well-characterized proges-
terone responsive MMTV promoter. The MMTV promoter
has been used extensively to study the mechanism of GR- and
PR-mediated transcription among others, and many of the
important factors involved in the activation of this promoter
are known. The MMTV promoter adopts a well-characterized
chromatin architecture with six rotationally phased nucleo-
somes termed A to F when stably integrated into the host
genome. Nucleosome A harbors the transcription start site,
while PR binding sites reside in the nucleosome B region and
the site of coactivator recruitment and SWI/SNF-induced nu-
cleosome remodeling (2). The 2963.1 cell line has the full-
length MMTV promoter integrated in the genome with the
CAT reporter that allows of analysis of transcription driven by
the MMTV promoter (45). Treatment of 2963.1 cells with
NAM leads to the loss of PR-mediated transcription of
MMTV much like that of endogenous genes (Fig. 3A). In
addition, the effect of NAM on PR-mediated transcription
activation of MMTV also occurs independently of SIRT1 and
PARP1, as shown by the siRNA knockdown–RT-PCR exper-
FIG. 3. NAM does not inhibit coactivator recruitment upon hormone treatment to the MMTV promoter. (A) T47D/2963.1 cells were treated
with the indicated amounts of NAM for 30 min, followed by treatment with R5020 (R) (10 nM) or with vehicle (EtOH) for 4 h. Total RNA was
harvested and analyzed by real-time RT-PCR with primers specific for MMTV or GAPDH as a control. The levels of transcripts for each gene
as determined by real-time PCR were normalized to those of GAPDH, and the value for the untreated control (?NAM, EtOH) was set to 1. The
error bars represent the standard error of the mean. (B) T47D/2963.1 cells were transfected with SIRT1, PARP1, both SIRT1 and PARP1 together
(S&P), or the scrambled nontargeting control (Control) siRNA as indicated for 48 h. The cells were then treated with 50 mM NAM or vehicle
(H2O) as ?NAM for 30 min, followed by treatment with R5020 (R) (10 nM) or with vehicle (EtOH) as ?R5020 control for 4 h. Total RNA
harvested from the siRNA-treated samples was analyzed by real-time RT-PCR with primers specific for MMTV. The levels of transcripts for each
of the genes were normalized against that of GAPDH, and the value for the untreated control (scrambled siRNA, ?NAM, ?R5020) was set to
1. The error bars represent the standard error of the mean. (C) T47/D/2963.1 cells were treated with 50 mM NAM or vehicle (H2O) as ?NAM
for 30 min, followed by treatment with R5020 (R) (10 nM) or vehicle (EtOH) as ?R5020 control for 1 h. The ChIP assay was performed using
antibodies against the indicated proteins. Nonspecific immunoglobulin G (IgG) (N.S. IgG) was used as background control. The graphs represent
the quantitation of real-time PCR results using primers specific to the promoter region (nucleosome B) of the MMTV-LTR. The error bars
represent the standard error of the mean. The BRG1 ChIP data were subjected to mixed-effects analysis of variance to determine statistically
significant differences between ?R5020 and ?R5020 values for BRG1 occupency. *, P ? 0.001; **, P ? 0.005.
34 AOYAGI AND ARCHERMOL. CELL. BIOL.
iment performed and analyzed identically to those of the en-
dogenous PR-regulated genes (Fig. 3B).
After we established that the MMTV transcription in re-
sponse to NAM is comparable to those of the endogenous
genes, we performed ChIP experiments to monitor the pro-
moter association of the PR and a transcription factor nuclear
factor 1 (NF1) and coactivators BRG1, CBP, and SRC3, all
known to be important for efficient hormone-induced activa-
tion of MMTV (2, 38). The ChIP experiments were performed
by treating 2963.1 cells with or without 30 min of NAM, fol-
lowed by 1 h with or without R5020, and the results of the
DNA precipitated with the indicated antibodies were analyzed
by real-time PCR using MMTV promoter primers (Fig. 3C)
and GAPDH gene primers as a background negative control
(data not shown). The results of the ChIP experiments show
that the ability of the PR to associate with the MMTV pro-
moter in a hormone-dependent manner is not compromised in
the presence of NAM. Coactivators BRG1 of the SWI/SNF
complex, the histone acetyltransferase CBP, p160/SRC family
of coactivator SRC3, and the transcription factor NF1 were
also efficiently recruited to the promoter in a hormone-depen-
dent manner in the presence of NAM (Fig. 3C).
One of the hallmarks of MMTV activation is the remodeling
of chromatin at the nucleosome B (promoter) region after
recruitment of the SWI/SNF complex (21, 58). It is possible,
however, that while the recruitment of BRG1, the ATPase
catalytic subunit of SWI/SNF, and other coactivators is not
compromised by NAM treatment (Fig. 3C), the efficiency of
the hormone-dependent increase in the accessibility of pro-
moter chromatin region diminishes in the presence of NAM.
To evaluate the extent of the chromatin accessibility of the
MMTV promoter, a restriction enzyme hypersensitivity assay
was performed using the SstI enzyme. The SstI enzyme cleav-
age site resides within the nucleosome B region of the MMTV
promoter (Fig. 4A), and the accessibility of this cleavage site
was monitored upon treatment with NAM and R5020. Isolated
nuclei from 2963.1 cells treated with or without NAM for 30
min, followed by 1 h with or without R5020, were digested with
the SstI enzyme, followed by digestion of the isolated DNA
with the HaeIII enzyme to completion for quantitation control.
The extent of SstI cleavage was monitored by reiterative PCR
analysis. Consistent with previous work, 2963.1 cells demon-
strate constitutive hypersensitivity at the SstI site in the ab-
sence of R5020 (Fig. 4B, lane 1) (45), which remains accessible
in the presence of NAM (Fig. 4B, lane 3). Upon hormone
treatment, nucleosome B hypersensitivity increases slightly, al-
though the effect is modest due to the constitutively open
nature of nucleosome B in the absence of hormone in this cell
line (Fig. 4B, compare lanes 1 and 2). This hormone-depen-
dent increase in hypersensitivity is maintained in the presence
of NAM after R5020 treatment (Fig. 4B, compare lanes 3 and
4). The analysis of MMTV nucleosome B remodeling was also
extended to the A1-2 cell line, which also has MMTV pro-
moter stably integrated in the genome with a luciferase
reporter (47). The MMTV in the A1-2 cell line is highly re-
sponsive to treatment with Dex. Much like the endogenous
GR-responsive genes, the induction of GR-dependent MMTV
transcription is inhibited in the presence of NAM (Fig. 1B and
data not shown). Unlike the 2963.1 cell line, the nucleosome B
region in the A1-2 cell line is not constitutively hypersensitive
and remains relatively inaccessible to SstI digestion, as ex-
pected (Fig. 4C, lane 1). Since the MMTV promoter in this
particular cell line is not efficiently induced by PR, changes in
chromatin accessibility were assessed after Dex treatment (3).
Upon Dex treatment, the accessibility at the SstI increases
significantly (Fig. 4C, lane 2). This increase in nucleosome B
accessibility upon hormone treatment occurs just as efficiently
in A1-2 cells treated with NAM as in those that were not
exposed to NAM (Fig. 4C, compare lanes 2 and 4).
In addition, ChIP experiments were performed using acety-
lated histone H3 and H4 antibodies to determine whether the
hormone-dependent chromatin modification such as histone
acetylation that is thought to contribute to the increased ac-
cessibility of chromatin is altered by NAM. ChIP experiments
were performed after treatment of 2963.1 cells with or without
FIG. 4. Chromatin remodeling of the MMTV promoter upon hor-
mone treatment is not inhibited by NAM. (A) Schematic of the prox-
imal MMTV promoter representing the hormone-sensitive nucleo-
some B region, restriction enzyme sites, and the primer (Oligo-22)
used for PCR analysis. T47/D/2963.1 (B) and T47D/A1-2 (C) cells
were treated with 50 mM NAM or vehicle (H2O) as ?NAM for 30
min, followed by treatment with R5020 (R) (10 nM), Dex (100 nM), or
vehicle (EtOH) as ?R5020 control for 1 h, as indicated. The nuclei
were harvested and digested with SstI in vivo. After genomic DNA
purification, all of the samples were digested to completion with
HaeIII for 2963.1 or BamHI for A1-2 cells in vitro as an internal
standard for the reiterative primer extension analysis using a
labeled primer (Oligo-22). The purified primer extension products
were separated on a 6% denaturing polyacrylamide gel, followed by
exposure to a phosphorimager screen. (D) T47/D/2963.1 cells were
treated with 50 mM NAM or vehicle (H2O) as ?NAM for 30 min,
followed by treatment with R5020 (R) (10 nM) or vehicle (EtOH) as
?R5020 control for 15 min. A ChIP assay was performed using anti-
bodies against either the pan-acetylated histone H3 or the pan-acety-
lated histone H4 as indicated. Nonspecific IgG (N.S. IgG) was used as
a background control. The graphs represent the quantitation of real-
time PCR results using primers specific to the promoter region (nu-
cleosome B) of the MMTV-LTR. The error bars represent the
standard error of the mean.
VOL. 28, 2008NICOTINAMIDE AND PR-MEDIATED TRANSCRIPTION35
NAM for 30 min, followed by 15 min of R5020, when the
histone acetylation levels were determined to be maximal in
our previous work or EtOH control (1). The ChIP results
demonstrate that the hormone-dependent increases in the pro-
moter histone acetylation levels are not altered upon NAM
treatment (Fig. 4D). These restriction enzyme hypersensitivity
and ChIP assays demonstrate that NAM does not inhibit tran-
scription by inhibiting the remodeling of the MMTV promoter
upon treatment with hormone.
NAM inhibits the PR-mediated transcription PIC assembly.
The ChIP and restriction enzyme hypersensitivity assays per-
formed thus far indicate that NAM does not suppress PR-
mediated transcription by inhibiting the recruitment of the
receptor and coactivators or chromatin remodeling of the pro-
moter. In order to determine whether NAM inhibits the re-
cruitment of the RNAPII machinery, ChIP assays were per-
formed to monitor RNAPII and TATA-binding protein (TBP)
of the TFIID complex recruitment after treatment of 2963.1
cells with or without NAM, followed by R5020 exposure. The
ChIP results show that, unlike the various coactivators that are
recruited to the promoter, both RNAPII and TBP are inhib-
ited from associating with the transcription start site region
(nucleosome A) (Fig. 5A). This raises the intriguing possibility
that NAM inhibits the communication between the activator
(PR) and the RNAPII transcription machinery. We postulated
that perhaps NAM inhibits the recruitment of the Mediator
complex by PR which is required for activation of many RNAPII-
regulated genes (5, 40, 57). To test this idea, ChIP experiments
were performed with antibodies to two of the Mediator com-
plex subunits MED1 and MED14. ChIP experiments per-
formed with or without NAM in the presence or absence of
hormone R5020 demonstrates that the hormone-dependent
recruitment of the Mediator complex, as assessed through
MED1 and MED14 subunits, is inhibited by NAM (Fig. 5B).
The exclusion of RNAPII, TBP, and Mediator from the
MMTV promoter is not due to changes in expression levels
upon NAM treatment, as determined by Western blot analysis
of whole-cell extracts (Fig. 5C). These results show that NAM
inhibits PR-mediated transcription by preventing the transcrip-
tion PIC to form, possibly due to the loss of recruitment of the
NAM-resistant gene HEF1 does not require MED1 and
MED14 for gene activation. The Mediator complex is thought
to play a critical role in bridging the interaction between the
transcription activator and the RNAPII machinery and for the
transcription of many of the RNAPII transcribed genes (5, 15,
35, 57). However, there are some genes that do not require
Mediator complex for transcription activation. For example,
different GR-responsive genes have been shown to require
different subunits of the Mediator complex for activation. For
example, the GILZ gene in the osteosarcoma cell line U2OS
was shown by siRNA knockdown experiments to require nei-
ther MED1 nor MED14 subunits of the Mediator complex
(14). We postulated that if NAM affects transcription at the
Mediator recruitment step, genes that do not require the Me-
diator complex for activation will not be affected by NAM. A
screen of additional PR- and GR-inducible genes identified the
HEF1 gene as Dex and R5020 inducible but unaffected by the
NAM treatment (Fig. 6A).
We predicted that the NAM-resistant HEF1 gene does not
require the Mediator complex for transcription activation. To
test this idea, both MED1 and MED14 were knocked down
simultaneously in the A1-2 cell line by the use of siRNA to
circumvent the possibility that one of the subunits may com-
pensate for the other during hormone-induced transcription.
The level of knockdown achieved for both MED1 and MED14
protein levels after 48 h of siRNA treatment compared to
treatment with scrambled nontargeted siRNA control were
achieved as demonstrated on the Western blot of prepared
whole-cell extracts in R5020 treated and untreated cells (Fig.
6B). After siRNA transfection, the cells were treated with
R5020 or vehicle (EtOH). RT-PCR analysis of the isolated
RNA shows that the R5020-induced transcription of CEBP/?,
SGK, and EZF genes is decreased after MED1 and MED14
knockdown (Fig. 6C). In contrast, the HEF1 gene transcription
FIG. 5. NAM inhibits the formation of PR-mediated PIC. T47/D/
2963.1 cells were treated with 50 mM NAM or vehicle (H2O) as
?NAM for 30 min, followed by treatment with R5020 (R) (10 nM) or
vehicle (EtOH) as a ?R5020 control for 1 h. (A) The ChIP assay was
performed using antibodies against RNAPII or TBP as indicated.
Nonspecific IgG (N.S. IgG) was used as a background control. The
graphs represent the quantitation of real-time PCR results using prim-
ers specific to the transcription start site region (nucleosome A) of the
MMTV-LTR. The error bars represent the standard error of the mean.
(B) The ChIP assay was performed with antibodies to MED1 or
MED14 as indicated. Nonspecific IgG (N.S. IgG) was used as a back-
ground control. The graphs represent the quantitation of real-time
PCR results using primers specific to the transcription start site region
(nucleosome B) of the MMTV-LTR. The error bars represent the
standard error of the mean. (C) Cellular levels of proteins as indicated
in the figure were determined by Western blot analysis of whole-cell
extracts derived from T47D/2963.1 treated as described above.
?-Tubulin was probed as a loading control.
36 AOYAGI AND ARCHERMOL. CELL. BIOL.
is completely unaffected by the decreased expression of MED1
and MED14, as predicted (Fig. 6C). These siRNA experiments
demonstrate that the hormone-dependent activation of the
HEF1 gene does not require the Mediator complex and is
therefore unaffected by NAM treatment, suggesting that NAM
inhibition of PR-mediated transcription occurs at the Mediator
Transcription is a complex process involving multiple en-
zymes and signaling pathways that intersect to regulate gene
expression. In recent studies we have described the dynamic
changes in histone acetylation and deacetylation that accom-
pany progesterone-induced transcription. These changes were
linked to the occupancy of canonical histone deacetylases
HDAC1 and HADC3 at the promoter (1). In the present study
we focused on the sirtuin family of proteins and the roles they
may play in PR-mediated transcription by analyzing the impact
of NAM on PR-mediated transcription in T47D human breast
cancer cells (2963.1 and A1-2 cells) (Fig. 1). There are seven
homologs of sirtuins, SIRT1 to SIRT7, with SIRT1 being by far
the most studied and the most closely linked to gene regulation
(23). SIRT1 deacetylates not only histones (H1, H3, and H4)
(9, 27, 60) but also coactivators involved in NR-mediated tran-
scription such as p300 (10) and PGC1? (46, 54). In addition,
SIRT1 has been shown to interact with PPAR? and downregu-
late its activity (50). SIRT1 has also been shown to deacetylate
AR, and treatment with SIRT1 inhibitor, NAM, induced li-
gand-dependent AR-mediated transcription (22). Further,
while NAM is a known sirtuin inhibitor, it is also known to
inhibit the PAR activities of PARP family of proteins (4, 17,
37). PARP1, the best-characterized member of the PARP fam-
ily of proteins, has been closely linked to transcription via its
ability to alter chromatin structure, as well as to regulation of
Mediator complex activity (33, 48).
Our results demonstrate that just half an hour of treatment
with NAM prior to hormone treatment suppressed hormone-
dependent activation of PR- and GR-regulated genes in a
dose-dependent manner. However, the siRNA knockdown of
SIRT1 and PARP1 demonstrated that NAM inhibition of tran-
scription was occurring through neither of these two factors
(Fig. 2B). This was surprising given the known roles of SIRT1
and PARP1 in the regulation of transcription and suggests the
presence of a novel mechanism by which NAM (a commonly
used SIRT1 and PARP1 inhibitor) inhibits transcription. The
novel transcription regulating factor that is inhibited by NAM
may involve other members of the sirtuin proteins. SIRT2,
SIRT6, and SIRT7 can be found in the nucleus and play a role
in the deacetylation of histones during mitosis, base excision
repair, and RNA polymerase I transcription, respectively (20,
23, 44, 61). It is possible that in addition to their known roles,
they are part of the PR-mediated transcription process that is
disrupted by NAM. Although the functions of the other mem-
bers of the sirtuins that are found outside of the nucleus, such
as the mitochondria, are not well known (9, 43), as their roles
in biology are discovered one could envision how they may also
affect transcription through processes such as posttranslational
modification of transcription factors as they shuttle in and out
of the nucleus. The PARP family of proteins, as well as other
NAD?metabolizing factors such as cyclic-ADP-ribosyl cy-
clases and members of the NAD?salvage pathway, are also
possible targets of NAM, and the identification of such a factor
is under investigation (7, 39). While SIRT1 and PARP1 may
not be the target of NAM inhibition of PR-mediated transcrip-
FIG. 6. Differential requirement for Mediator subunits by PR reg-
ulated genes. (A) T47D/A1-2 cells were treated with the indicated
amounts of NAM or vehicle (H2O) for 30 min, followed by treatment
with either Dex (100 nM), R5020 (10 nM), or vehicle (EtOH) for 4 h
as indicated. Total RNA was harvested and analyzed by real-time
RT-PCR with primers specific for the HEF1 gene or GAPDH as a
control. The levels of transcripts for the HEF1 gene as determined by
real-time PCR were normalized to those of GAPDH, and the value for
the untreated control (0 mM NAM, EtOH) was set to 1. The error bars
represent the standard error of the mean. (B) T47D/A1-2 cells were
transfected with MED1 and MED14 siRNA together or the scrambled
nontargeting control (Control) siRNA for 48 h. The cells were then
treated with R5020 (10 nM) or with vehicle (EtOH) as ?R5020 control
for 4 h. The cellular levels of MED1, PARP1, and PR were determined
by Western blot analysis of whole-cell extracts derived from the
siRNA-treated cells. ?-Tubulin was probed as a loading control.
(C) Total RNA harvested from the siRNA-treated T47D/A1-2 samples
was analyzed by real-time RT-PCR with primers specific for the indi-
cated genes. The levels of transcripts for each of the genes were
normalized against that of GAPDH, and the value for the untreated
control (scrambled siRNA, ?R5020) was set to 1. The error bars
represent the standard error of the mean.
VOL. 28, 2008 NICOTINAMIDE AND PR-MEDIATED TRANSCRIPTION37
tion under our conditions, it still remains to be seen whether
they have a role in regulating the gene expression of some
genes such as EZF and SGK which show decreased expression
upon SIRT1 and PARP1 knockdown (Fig. 2B).
To further dissect the mechanism by which NAM inhibits
PR-mediated transcription, ChIP experiments were performed
using the MMTV as a model PR-activated promoter. Because
the NR target promoters are regulated in the context of chro-
matin, the receptor must recruit various coactivators and ATP-
dependent chromatin remodeling complexes to render the
promoter DNA more accessible (24, 34, 55). The ChIP exper-
iments we performed demonstrate that the hormone-depen-
dent association of PR is not compromised (Fig. 3C). PR also
retains the ability to recruit various coactivators and allow the
promoter chromatin to be more accessible as determined by
ChIP and restriction enzyme hypersensitivity assays in the
presence of NAM (Fig. 3C and 4). Interestingly, our ChIP
results show that inhibition of PR-mediated transcription oc-
curs at the step of PIC assembly. This suggests that NAM
inhibits the assembly of basal transcription machinery after
chromatin remodeling of the promoter and that the commu-
nication between PR-mediated chromatin remodeling events
and the assembly of basal transcription machinery is disrupted.
In particular, NAM inhibition of PIC assembly appears to be
occurring at the Mediator complex recruitment step. The Me-
diator complex has been shown to facilitate the recruitment of
TFIID by binding cooperatively with it on promoters and to
facilitate the recruitment of RNAPII through the interaction
with CTD of RNAPII and transcription activators, thereby
acting as a “bridge” to connect the transcription factor activi-
ties with the PIC machinery (5, 28, 35). The observations from
the siRNA knockdown experiments of subunits of Mediator
(MED1 and MED14) are consistent with a clear role for the
Mediator complex in vital steps in the assembly of basal tran-
scription machinery, and NAM most likely interferes with this
step, thereby leading to the inhibition of PR-mediated tran-
scription (Fig. 6). NAM could potentially directly or through
other factors affect either or both the PR or the Mediator
complex itself by altering posttranslational modifications or
inducing conformational changes, which leads to the inhibition
of Mediator complex recruitment to target genes. In consider-
ing these possibilities, it is also important to determine the fate
of NAM and whether this leads to changes in nuclear NAD?
concentration within our experimental parameters since the
enzymes involved in the NAD?consumption (by proteins such
as SIRT1 and PARP1) and regeneration by the NAD?salvage
pathway are present in the nucleus (39, 64). This is an impor-
tant factor to consider when we further characterize the mech-
anism by which NAM inhibits the recruitment of the Mediator
complex. NAM will most probably affect transcription by other
steroid hormone receptors such as ER and AR in a similar
manner as we have demonstrated for PR, given the similarities
in the mechanism of transcription activation involving the Me-
diator complex. Depending on the exact nature of NAM inhi-
bition of Mediator recruitment and whether this step takes
place in the activation of genes by other families of transcrip-
tion activators, it remains to be seen whether NAM has a
similar effect on a wide range of transcription pathways.
We demonstrate here that transcription factor recruitment
and promoter chromatin remodeling events can be uncoupled
from transcription activation. This NAM-mediated inhibition
is independent of SIRT1 and PARP1. This indicates that NAM
blocks the regulation of an as-yet-unknown but critical mech-
anism or pathway required for the chromatin remodeling
events to be translated into PIC formation and the activation
of gene expression.
We thank Paul Wade, Xioling Li, Pratibha Hebbar, and Harriet
Kinyamu (NIEHS) for critical reviews for the manuscript; Micheal
Garabedian (NYU School of Medicine) for the MED1 and MED14
antibodies; and Grace Kissling (NIEHS) for the statistical analyses.
This research was supported by the Intramural Research Program of
the NIH and NIEHS.
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