The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates.
ABSTRACT To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.
- SourceAvailable from: PubMed CentralAsian Journal of Andrology 06/2014; · 2.53 Impact Factor
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ABSTRACT: The acidic (leucine-rich) nuclear phosphoprotein 32 kDa (ANP32) family is composed of small, evolutionarily conserved proteins characterized by an N-terminal leucine- rich repeat domain and a C-terminal low-complexity acidic region. The mammalian family members (ANP32A, ANP32B, and ANP32E) are ascribed physiologically diverse functions including chromatin modification and remodelling, apo- ptotic caspase modulation, protein phosphatase inhibition, as well as regulation of intracellular transport. In addition to reviewing the widespread literature on the topic, we present a concept of the ANP32s as having a whip-like structure. We also present hypotheses that ANP32C and other intronless sequences should not currently be considered bona fide family members, that their disparate necessity in develop- ment may be due to compensatory mechanisms, that their contrasting roles in cancer are likely context-dependent, along with an underlying hypothesis that ANP32s represent an important node of physiological regulation by virtue of their diverse biochemical activities.BioEssays 11/2014; 36(11). · 4.84 Impact Factor
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ABSTRACT: SET is a multifunctional protein involved in regulating many biological processes of the cell cycle. It is also a regulator of steroidogenesis in the ovary. However, the expression of SET protein in testis, and its function, still remains ambiguous. In this study, we observed the expression of SET in the testes of mice at different developmental stages, and have discussed its potential function in regulating spermatogenesis and androgen production. Forty-eight male mice at different developmental stages (1 week old as the infancy group; 4 weeks old as the prepubertal group; 12 weeks old as the adult group; over 12 months old as the ageing group) were used. Cellular location of SET protein in the testes was observed by immuno-histochemistry. Expression levels of Set mRNA and SET protein were analyzed by quantitative polymerase chain reaction and Western blotting. SET protein was expressed in spermatogonial cells and spermatocytes; the highest level was mainly in haploid and tetraploid cells of the prepubertal and adult groups, and Leydig cells of the adult and ageing groups. There was a low expression in Sertoli cells. Expression of Set mRNA in the prepubertal group was significantly higher than that in the adult group (P < 0.05), while expression of SET protein was at the highest level in the adult group (P < 0.05). SET protein is mainly expressed in spermatogonial cells and spermatocytes, and poorly expressed in Sertoli cells, suggesting that it is involved in spermatogenesis. Expression of SET protein in Leydig cells suggests a possible role in steroidogenesis.Asian Journal of Andrology 06/2014; · 2.53 Impact Factor
MOLECULAR AND CELLULAR BIOLOGY, Jan. 2005, p. 797–807
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 2
The Histone Chaperone TAF-I/SET/INHAT Is Required for
Transcription In Vitro of Chromatin Templates
Matthew J. Gamble,1,2Hediye Erdjument-Bromage,1Paul Tempst,1
Leonard P. Freedman,3† and Robert P. Fisher1*
Molecular Biology Program1and Cell Biology Program,3Memorial Sloan-Kettering Cancer Center, and
Programs in Biochemistry, Cell and Molecular Biology, Cornell University Graduate School of
Medical Sciences,2New York, New York
Received 23 July 2004/Returned for modification 2 September 2004/Accepted 18 October 2004
To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a
mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor
of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of
chromatin transcription responsive to the vitamin D3receptor or to Gal4-VP16. TAF-I associates with chro-
matin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA
templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The
chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated
temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I
during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added
after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is
required to facilitate transcription at a step after chromatin assembly but before transcript elongation.
The responsibility for transcribing protein-coding genes in
eukaryotes belongs, with very rare exceptions, to RNA poly-
merase II (Pol II). To fulfill that role in vivo, Pol II and its
accessory proteins must not only recognize transcriptional pro-
moters accurately but must also modify the transcriptional
repertoire appropriately in response to internal and external
signals. Furthermore, Pol II must perform these tasks on DNA
within a chromatin structure that is generally repressive. Bio-
chemical analyses over the last 2 decades have identified a
myriad of accessory factors required to initiate transcription
accurately and to respond to DNA-binding transcriptional ac-
tivators. It is now possible to reconstitute transcription respon-
sive to activators in vitro with recombinant and highly purified
components (21). However, the minimal system sufficient to
transcribe naked DNA templates fails to support activator-
dependent transcription of templates assembled into chroma-
The basic unit of chromatin is the nucleosome, an octamer
of core histones (H2A, H2B, H3, and H4) around which 147 bp
of DNA wrap with a physiologic repeat length averaging 200
bp. There are several reasons why DNA assembled into chro-
matin requires additional factors, not needed on naked tem-
plates, to support activator-dependent transcription. First, the
presence of nucleosomes on promoter DNA can inhibit re-
cruitment of the basal transcriptional machinery to form a
preinitiation complex (20). Second, chromatin structure in
transcribed regions poses a physical barrier to elongation by
Pol II (41). Finally, nucleosomes are substrates for covalent
modifications by a variety of enzymes that can facilitate or
repress transcription of nearby genes at least in part by pro-
viding docking platforms for coactivators or corepressors (17).
The earliest enzymatic systems capable of assembling cor-
rectly spaced nucleosomal arrays onto cloned DNA—a prereq-
uisite for defining the requirements for chromatin-templated
transcription—consisted of extracts derived from early em-
bryos, purified core histones, and ATP (3, 12). A drawback of
these systems, however, is that crude embryonic extracts may
themselves contain the unidentified factors required for chro-
matin-based transcription, necessitating purification of the
chromatin after assembly.
Completely recombinant chromatin assembly systems have
also been described that generally contain an ATP-dependent
chromatin remodeling factor of the ISWI family and a histone
chaperone (23, 24). Transcription of chromatin made with
recombinant assembly systems depends, however, on a crude
nuclear extract. More defined systems are not competent to
transcribe chromatin templates assembled with recombinant
proteins, indicating a requirement for additional factors.
To reveal those requirements, we fractionated a HeLa cell
nuclear extract capable of activated transcription on purified
chromatin templates. We identified and purified TAF-I/SET
(template-activating factor I/patient SE translocation), a mem-
ber of the nucleosome assembly protein (NAP) family of his-
tone chaperones, as a factor required to transcribe chromatin.
The requirement appears to be general; TAF-I potently stim-
ulated transcription driven by the 1,25(OH)2vitamin D3re-
ceptor (VDR), as well as by the GAL4-VP16 transactivator.
The related NAP-1 protein, which was used to assemble the
chromatin templates, can substitute for TAF-I in the transcrip-
tion reaction. Conversely, TAF-I can substitute for NAP-1 in
the assembly of chromatin. We show, however, that TAF-I
plays distinct roles in assembling and transcribing chromatin;
* Corresponding author. Mailing address: Molecular Biology Pro-
gram, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New
York, NY 10021. Phone: (212) 639-8912. Fax: (212) 717-3317. E-mail:
† Present address: Department of Molecular Endocrinology, Merck
Research Laboratories, West Point, PA 19486.
transcription of chromatin assembled with TAF-I instead of
NAP-1, or of NAP-1-assembled chromatin preincubated with
TAF-I, is still dependent on the presence of TAF-I during the
polymerization reaction. Although TAF-I acts after chromatin
is assembled, it is required prior to the elongation phase of
the transcription cycle, distinguishing it mechanistically from
FACT, a factor required for Pol II to overcome nucleosomal
barriers to elongation (36). Thus, we have uncovered a novel
function for the NAP family of histone chaperones at an early
step in transcriptional activation on chromatin templates.
MATERIALS AND METHODS
Expression and purification of recombinant proteins. Amino-terminally His-
tagged TAF-I bacterial expression vectors were constructed by cloning the TAF-
I?, TAF-I?, or TAF-I?(1–225) open reading frame into pET-14b. A hemagglu-
tinin (HA)-tagged version of TAF-I? was made by cloning the HA sequence
upstream of the His tag in pET-14b. FLAG-tagged TAF-I? was made by replac-
ing the His tag in pET-28a with the FLAG sequence upstream of the TAF-I?
open reading frame. The His–TAF-I, HA–His–TAF-I, and Gal4-VP16 (14) con-
structs were expressed in Escherichia coli and purified by sequential cobalt
affinity and Q anion-exchange chromatography. FLAG–TAF-I? was purified by
anti-FLAG immunoaffinity chromatography. The FLAG–TAF-I?/His–TAF-I?
dimer was coexpressed in E. coli and purified by sequential cobalt affinity and
anti-FLAG immunoaffinity chromatography. rVDR and rRXR were overex-
pressed in Sf9 cells and purified as previously described (22). Recombinant
ATP-dependent chromatin assembly factor (ACF) complex (rACF) and recom-
binant NAP-1 (rNAP-1) were expressed and purified as previously described
(15). rTFIIA was purified as previously described (13).
Chromatin assembly and micrococcal nuclease digestion. A plasmid contain-
ing four vitamin D3response elements (VDREs) upstream of an adenovirus E1B
core promoter fused to a cat reporter gene, 4 ? VDRE B/INR, or a plasmid
containing five Gal4-binding sites upstream of an adenovirus E4 promoter,
pGEIO (14), was used as the substrate for chromatin assembly. Chromatin
assembly and micrococcal nuclease digestion were performed essentially as pre-
viously described (15), except that supercoiled plasmid DNA was used instead of
topoisomerase I-relaxed DNA. The final reaction conditions were as follows:
4.42 ng of plasmid DNA per ?l, 16 nM rACF, 0.9 ?M rNAP-1, 13 ?g of purified
Drosophila core histones per ?l, 3.4 mM ATP, and an ATP regeneration system.
In the case of chromatin assembled with rTAF-I?, rNAP-1 was omitted from the
assembly reaction mixture. In the case of chromatin assembled with S190 extract,
2 ?g of S190 extract per ?l was added to the reaction mixture instead of rACF
and rNAP-1. After assembly for 3 h at 27°C, the chromatin was aliquoted, frozen
in liquid N2, and stored at ?80°C for use in micrococcal nuclease digestion or
HeLa nuclear extract preparation and fractionation. Nuclear extracts were
typically prepared from 100 liters of HeLa cells harvested at a density of 5 ? 105
cells per ml. Nuclear extracts were prepared essentially as previously described
(8), except that KCl replaced NaCl in all buffers and a protease inhibitor cocktail
(P8340; Sigma) was included during the homogenization step. The nuclear ex-
tract was dialyzed against 20 mM HEPES (pH 7.9)–0.2 mM EDTA–0.3 M
KCl–20% glycerol–1 mM dithiothreitol (DTT).
All fractionation was done at 4°C in HEG (20 mM HEPES [pH 7.9], 0.2 mM
EDTA, 10% glycerol, 1 mM DTT) containing the indicated concentrations of
KCl. HeLa nuclear extract (600 mg) was loaded onto a 50-ml phosphocellulose
P-11 (Whatman) column equilibrated at 0.3 M KCl. The PC1 fraction used as the
source of general transcription factors was generated by eluting the column with
HEG–1 M KCl. The PC1 fraction was then dialyzed against HEG–50 mM
KCl–20% glycerol. The unbound fraction (PC.3) was the starting point for the
purification of Pol II and TAF-I/SET, as described in Results. The column
buffers were supplemented with 0.1% Tween 20 for all steps after DEAE chro-
Transcription assays. Transcription assays were performed essentially as pre-
viously described (37), except that TFIIA was added in recombinant form and
the PC1 fraction was the source of basal transcription machinery. The 25-?l
reaction mixture contained 31.7 ng of template DNA assembled into chromatin,
10 nM RXR, 30 nM VDR, 3 ?g of PC1, 27 ng of rTFIIA, 50 mM KCl, 20 mM
HEPES, 2% polyvinyl alcohol, 10% glycerol, 2.8 ?M acetyl coenzyme A, 2.7 mM
MgCl2, and 1.25 mM nucleoside triphosphate. Unless otherwise indicated, 1 ?M
vitamin D3was added to all reaction mixtures containing VDR. All of the
transcription reaction mixtures in Fig. 5 to 8 contain 0.78 ?g of immunopurified
Pol II. Other factors were added as indicated. Where stated, chromatin was
purified away from unbound factors by chromatography on a 500-?l S300 spin
column equilibrated in R buffer (10 mM HEPES [pH 7.6], 10 mM KCl, 1.5 mM
MgCl2, 0.5 mM EGTA, 10% glycerol, 10 mM ?-glycerophosphate, 0.25 mM
phenylmethylsulfonyl fluoride, 1 mM DTT). Typically, 80 ?l of chromatin was
loaded on the column and the purified chromatin was collected by centrifugation
in a tabletop centrifuge. Transcription was allowed to proceed at 30°C for 1 h
after nucleoside triphosphate addition. The products were analyzed by primer
extension and 6% denaturing gel electrophoresis. The gel was dried and exposed
to film at ?80°C or quantified with a Phosphorimager.
Transcription elongation assays were set up essentially as described above,
with the following exceptions. The transcription reactions were initiated by
addition of ATP, UTP, and GTP to a 1 mM final concentration and 5 ?Ci of
[?-32P]CTP and incubated for 2 min at 30°C. The reaction mixture was then
supplemented with unlabeled CTP to a final concentration of 1 mM. Where
indicated, TAF-I? was added at 3 ?M either before the pulse-labeling or 1 min
after the unlabeled CTP.
Protein identification. Proteins excised from gels were digested with trypsin,
the mixtures were fractionated on a Poros 50 R2 RP microtip, and the resulting
peptide pools were analyzed by matrix-assisted laser desorption ionization–time
of flight mass spectrometry (MS) with a BRUKER UltraFlex TOF/TOF instru-
ment (Bruker Daltonics, Bremen, Germany) as previously described (9, 45).
Selected experimental masses (m/z) were taken to search the human segment of
the National Center for Biotechnology Information (Bethesda, Md.) nonredun-
dant protein database (?108,000 entries) with the PeptideSearch algorithm
(Matthias Mann, Southern Denmark University, Odense, Denmark), with a mass
accuracy restriction of better than 40 ppm and a maximum of one missed
cleavage site allowed per peptide. Mass spectrometric sequencing of selected
peptides was done by matrix-assisted laser desorption ionization–time of flight-
time of flight (MS/MS) analysis on the same prepared samples with the UltraFlex
instrument in LIFT mode. Fragment ion spectra were taken to search the Na-
tional Center for Biotechnology Information nonredundant protein database
with the MASCOT MS/MS Ion Search program (Matrix Science Ltd., London,
United Kingdom). Any identification thus obtained was verified by comparing
the computer-generated fragment ion series of the predicted tryptic peptide with
the experimental MS/MS data.
Chromatin immunoprecipitation in vitro. Transcription reactions were al-
lowed to proceed for 30 min in the presence of 3 ?M His–TAF-I? or HA–His–
TAF-I? and then diluted to 200 ?l with HEG–50 mM KCl–0.1% Tween 20.
Immunoprecipitations were then performed with 5 ?l of HA antibody (16B12
ascites fluid; Covance) and 15 ?l of protein G Sepharose (Amersham). The
beads were washed four times with HEG–150 mM KCl–0.1% Tween 20 and once
with HEG–50 mM NaCl. The beads were then eluted with 200 ?l of 1% sodium
dodecyl sulfate (SDS)–50 mM NaCl. The samples were digested with proteinase
K (Sigma) and subjected to phenol-chloroform extraction and ethanol precipi-
tation. The amount of DNA recovered was quantified by PCR with primers
flanking the promoter.
HA–TAF-I protein immunoprecipitation. The interaction of TAF-I and en-
dogenous SNF2H was detected by incubating the indicated amounts of His–
TAF-I? or HA–His–TAF-I? with PC1 for 30 min at room temperature. The
reaction mixture was then rocked in the presence of 15 ?l of protein G beads
(Amersham) covalently coupled to HA antibody (16B12; Covance) at room
temperature for 1 h. The beads were washed four times with HEG–150 mM
KCl–0.1% Tween 20 and once with HEG–50 mM NaCl. The beads were eluted
in SDS gel loading buffer, and bound proteins were separated by SDS–6%
polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose mem-
brane, and detected with anti-hSNF2 (1B9/D12; Upstate Biotechnology).
Anti-SNF2 depletion of PC1 fraction. Ten micrograms of anti-SNF2 (1B9/D12;
Upstate Biotechnology) was prebound to 20 ?l of protein G Sepharose. The
anti-SNF2-coupled beads or control protein G beads were mixed with 300 ?g of
PC1 fraction and incubated at 4°C with agitation for 2 h. The supernatants from
the control beads (mock-depleted PC1) and anti-SNF2 beads (SNF2H-depleted
PC1) were collected and used in transcription reaction mixtures. The depletion
was monitored by immunoblotting with the anti-SNF2 antibody.
Identification of a fraction required for VDR-mediated tran-
scription on chromatin. We set out to define the components
of a HeLa cell nuclear extract required to support transcription
on a chromatin template. When we incubated a reporter plas-
798GAMBLE ET AL.MOL. CELL. BIOL.
mid containing four VDREs upstream of an adenovirus E1B
minimal core promoter with purified core histones and a Dro-
sophila embryonic S190 extract, nucleosomes were deposited
in a physiologically spaced array, as revealed by limited micro-
coccal nuclease digestion (Fig. 1A, left). In the presence of
VDR, a fractionated transcription system derived from the
phosphocellulose-bound fraction of a HeLa nuclear extract
was capable of ligand-responsive transcription from crude
chromatin assembled in the S190 extract (Fig. 1C, lanes 1 and
2). However, when the chromatin was partially purified from
the S190 extract by gel filtration, ligand-dependent transcrip-
tion was nearly abolished (Fig. 1C, lanes 5 and 6). Therefore,
components of the S190 extract not stably incorporated into
the chromatin are apparently required for efficient transcrip-
tion. Transcription on the purified chromatin was restored by
adding the phosphocellulose-unbound fraction of the nuclear
extract (PC.3) to the reaction mixture (Fig. 1C, lanes 7 and 8),
indicating that PC.3 contained an activity or activities required
for VDR-activated transcription on chromatin templates. We
next fractionated the PC.3 fraction by DEAE anion-exchange
chromatography; transcription-stimulatory activity eluted in a
single peak at ?330 mM KCl, a fraction we designated DE330
Because the S190 extract contains potentially confounding
activities capable of stimulating chromatin transcription, we
next tested the ability of the DE330 fraction to stimulate tran-
scription on chromatin assembled in a system consisting of
purified core histones and recombinant NAP-1 and ACF (a
complex of the catalytic subunit ISWI and the accessory factor
Acf1). Chromatin assembled in the recombinant system also
contained well-spaced nucleosomes (Fig. 1A, right). Just as
PC.3 was required to transcribe partially purified chromatin
assembled in the S190 extract, DE330 was required for tran-
scription from chromatin assembled in the recombinant system
(Fig. 1D), which was the source of the chromatin used in all
subsequent assays. We measured ?25-fold stimulation of ac-
tivated transcription in the presence of vitamin D3by the
DE330 fraction at the maximal concentration attainable in the
assay. Transcription in the absence of vitamin D3also in-
creased with increasing amounts of the DE330 fraction, but
there was an essentially constant ratio of activated-to-basal
transcription at all concentrations for which a signal could be
measured above the background in the absence of ligand, in-
dicating that the DE330 fraction facilitated but did not circum-
vent VDR-mediated activation of transcription on chromatin
Pol II cofractionates with chromatin transcription activity.
We further purified the stimulatory activity by Q Sepharose
anion-exchange and Superdex 200 gel filtration chromatogra-
phy (Fig. 2A). We observed a peak of activity in Superdex 200
fractions 20 to 22, corresponding to an apparent molecular
mass of ?500 kDa (Fig. 2C), although ?70% of the activity
was lost during the gel filtration step. Four polypeptides of 38,
22, 18, and 16 kDa cofractionated with the activity (Fig. 2D).
Mass spectrometric analysis identified the 38- and 22-kDa spe-
cies as the Rpb3 and Rpb7 subunits of Pol II, respectively.
Immunoblotting confirmed the copurification of Rpb1, the
largest subunit of Pol II, with transcription-stimulatory activity
(data not shown).
To determine whether the Pol II enzyme purified from the
DE330 fraction is necessary and/or sufficient to reconstitute
transcription on chromatin, we subjected the fraction to anti-
Rpb1 immunoaffinity chromatography (Fig. 3A). Pol II was
efficiently depleted from the DE330 fraction by passage over
an anti-Rpb1 monoclonal antibody (8WG16) column and
could be eluted with a peptide corresponding to the epitope:
the carboxyl-terminal domain (CTD) of Rpb1 (Fig. 3B and C).
The CTD peptide eluate contained highly purified Pol II, as
judged by silver staining after SDS-PAGE (Fig. 3B); we de-
FIG. 1. Identification of activities required for chromatin transcrip-
tion. (A) Titration of micrococcal nuclease in a limited digestion of
promoter-containing DNA assembled into chromatin with crude chro-
matin assembly extract (S190) or with a recombinant chromatin as-
sembly system (rACF and rNAP-1). (B) Fractionation of HeLa nuclear
extract. (C) VDR-mediated transcription reaction mixtures were pre-
pared with S190-assembled chromatin left unpurified (crude chroma-
tin) or purified over an S300 spin column (purified chromatin) in the
presence or absence of the PC.3 fraction (12 ?g) and vitamin D3as
indicated. (D) VDR-mediated transcription reaction mixtures were
prepared on chromatin assembled in the recombinant system in the
presence or absence of vitamin D3and buffer only or 0.25, 0.5, 0.75,
1.0, 1.5, or 2 ?l of the DE330 fraction, as indicated. The relative level
of transcription was quantified by Phosphorimager, with the maximal
level defined as 100 U, and is indicated below each lane.
VOL. 25, 2005 TAF-I IS REQUIRED FOR CHROMATIN TRANSCRIPTION799
tected all known subunits of Pol II in apparently equal stoichi-
ometry, as well as several high-molecular-weight polypeptides
that were apparently substoichiometric (Fig. 3B).
Depleting the DE330 fraction of Pol II nearly abolished
activity, indicating that this source of Pol II was indeed re-
quired for efficient transcription from chromatin templates
(Fig. 3D, compare lanes 2 and 3). Whether this represented a
quantitative effect of increasing the Pol II concentration rela-
tive to those of the other PC1 components or a specific re-
quirement for the form of Pol II that flows through phospho-
cellulose remains to be determined. The purified Pol II enzyme
was insufficient, however, to reconstitute the full activity of the
parent DE330 fraction. In fact, roughly six times more purified
Pol II, relative to the Pol II in the starting material, was re-
quired to produce equivalent transcription signals (Fig. 3D,
compare lanes 2 and 6).
TAF-I/SET is required for transcription from chromatin.
Because neither the anti-Rpb1 flowthrough fraction nor puri-
fied Pol II alone could fully reconstitute the activity of the
starting fraction, we performed mixing experiments. When we
added both the anti-Rpb1 flowthrough fraction and immuno-
purified Pol II, we observed full reconstitution of the input
activity. The stimulation of transcription by the two fractions
was more than additive (Fig. 3D, compare lanes 3 and 5 to lane
7), indicating that both Pol II and a distinct activity in the
anti-Rpb1 unbound fraction are required for chromatin-tem-
We further purified the activity in the anti-Rpb1 flow-
through fraction by Q Sepharose anion-exchange and Super-
dex 200 gel filtration chromatography (Fig. 4A). The activity in
the anti-Rpb1 flowthrough fraction was only detected in the
presence of immunopurified Pol II; we therefore assayed the
column fractions in the presence of saturating amounts of Pol
II. Under these conditions, we measured a ?100% yield during
gel filtration (Fig. 4B, upper part). Also, in contrast to the
sizing column run prior to depletion of Pol II (Fig. 2C), the
transcription-stimulatory activity migrated with an apparent
size of ?200 kDa (rather than ?500 kDa). Two major polypep-
tides of 44 and 42 kDa cofractionated with activity during gel
filtration (Fig. 4B, lower part). Mass spectrometric analysis
determined that both of these polypeptides were derived from
TAF-I/SET (hereafter referred to as TAF-I).
FIG. 2. Pol II copurifies with chromatin transcription activity. (A) Scheme for the further fractionation of PC.3. (B) VDR-mediated transcrip-
tion reaction mixtures were prepared with 1.5 ?l of the indicated DEAE fractions. The peak of activity eluted at ?330 mM KCl. (C) VDR-
mediated transcription reaction mixtures were prepared with 2 ?l of the indicated Superdex 200 fractions or 1 ?l of the load. (D) The Superdex
200 fractions were resolved by SDS–10% PAGE, and proteins were visualized by silver staining. Migration of polypeptides correlating with activity
is indicated by asterisks. Mass spectrometry identified two of these as Rpb3 and Rpb7, as indicated. The values on the left are molecular sizes in
kilodaltons. L, load; F, flowthrough.
800 GAMBLE ET AL.MOL. CELL. BIOL.
TAF-I was originally identified as the product of a gene
fused to the CAN gene in an acute undifferentiated myeloid leu-
kemia (43) and was first characterized biochemically as a cel-
lular factor required for the replication of purified adenovirus
core particles in vitro (31). TAF-I contains a central domain ho-
mologous to NAP-1 and other members of the NAP family of
histone chaperones. TAF-I exists in two isoforms generated by
variable splicing—TAF-I? and TAF-I?—that are identical ex-
cept for unique stretches of 37 and 24 amino acids, respec-
tively, at the amino terminus. Immunoblot assays with antibod-
ies specific for either TAF-I? or TAF-I? showed that both were
present in the peak fraction obtained by Superdex 200 chroma-
tography (Fig. 4C). We purified TAF-I? tagged with hexahisti-
dine (His) at the amino terminus and expressed in bacteria.
The recombinant TAF-I? (rTAF-I?) obtained was sufficient to
reconstitute the activity in the Superdex 200 peak fractions
(Fig. 4D), indicating that TAF-I is the only required factor in
those fractions and that TAF-I? suffices for full activity.
TAF-I is a chromatin-specific coactivator for multiple acti-
vators. As demonstrated above (Fig. 3D), the DE330 fraction
contained two components required to reconstitute chromatin-
templated transcription in vitro: Pol II and TAF-I. To deter-
mine the factor(s) in which chromatin specificity resides, we
tested the effect of each component, alone or in combination,
on transcription performed with naked or chromatin-assem-
bled DNA templates (Fig. 5A). The immunopurified Pol II
enzyme was capable of stimulating transcription by at least
10-fold on both chromatin and naked templates, regardless of
the presence of TAF-I?. On the other hand, rTAF-I? stimu-
lated transcription on chromatin by ?10-fold but had a less-
than-2-fold effect on transcription of naked DNA over the
range of Pol II concentrations tested. Thus, rTAF-I? prefer-
entially stimulated transcription of chromatin templates.
We next sought to determine whether TAF-I acts as a spe-
cific coactivator for nuclear receptors or plays a more general
role. We tested the effect of TAF-I on transcription activated
by Gal4-VP16—a fusion of the yeast Gal4 DNA-binding do-
main with the activation domain of the viral transactivator
VP16—of a chromatin template containing five Gal4-binding
sites upstream of the E4 viral promoter. The level of Gal4-
VP16-mediated transcription was enhanced about sixfold by
rTAF-I? (Fig. 5B). Therefore, stimulation of chromatin tran-
scription by TAF-I is not restricted to promoters activated by
nuclear receptors, suggesting a more general role in facilitating
Characterization of TAF-I in transcription. The TAF-I pro-
tein we purified from HeLa cells was a nearly stoichiometric
mixture of TAF-I? and TAF-I?. To examine the effect of
TAF-I? on chromatin transcription, TAF-I? was expressed in
bacteria and purified identically to rTAF-I?. rTAF-I? was also
able to stimulate chromatin transcription, albeit with a specific
activity more than twofold lower than that of TAF-I? (Fig. 6A).
TAF-I? and TAF-I? have been shown to form dimers in
extracts and when expressed in bacteria (32). To compare the
activity of TAF-I? with that of a presumptive TAF-I?/? dimer,
we constructed rTAF-I? in which the His tag was replaced with
a FLAG tag. We expressed FLAG–rTAF-I? together with
His–TAF-I? in E. coli and purified, by sequential cobalt and
FLAG affinity chromatography, a complex containing TAF-I?
and TAF-I? in equal proportions, consistent with a hetero-
dimer (Fig. 6B). In transcription reaction mixtures, the relative
activities of the three complexes were as follows: TAF-I?/
TAF-I? ? TAF-I? alone ? TAF-I? alone (Fig. 6C).
The acidic carboxyl terminus of TAF-I is important for sev-
eral of its previously reported functions (34, 39, 40). We ex-
pressed and purified a truncation mutant form that lacked the
52 amino acids corresponding to the acidic carboxyl terminus
[rTAF-I?(1–225)]. The truncated protein was completely in-
active in transcription, indicating that the carboxyl terminus is
required to stimulate transcription on chromatin (Fig. 6D, left
part). The rTAF-I?(1–225) mutant construct was also capable
of inhibiting the activity of wild-type TAF-I when added to the
same reaction mixture (Fig. 6D, right part).
Because TAF-I is closely related to NAP-1, we investigated
whether NAP-1 could also stimulate transcription. NAP-1 was
indeed capable of substituting for TAF-I but had about twofold
lower specific activity (Fig. 6E). Thus, stimulation of transcrip-
FIG. 3. Pol II is required but not sufficient to reconstitute the
chromatin transcription activity in the DE330 fraction. (A) Pol II
immunoaffinity chromatography scheme to purify Pol II from the
DE330 fraction. (B) Immunopurified Pol II was analyzed by SDS–12%
PAGE and silver staining. Pol II subunits are indicated at the right.
The values on the left are molecular sizes in kilodaltons. (C) Immu-
noblot, probed with anti-Rpb1 monoclonal antibody 8WG16, of equiv-
alent volumes of DE330, the anti-Rpb1 flowthrough fraction, and
immunopurified Pol II. (D) VDR-mediated transcription reaction mix-
tures prepared with buffer only (lane 1); 2 ?l of DE330 (lane 2); 2 ?l
of the anti-Rpb1 flowthrough fraction (lane 3); 1, 2, or 4 ?l of immu-
nopurified Pol II, corresponding to roughly 1.5, 3, or 6 times the
amount of Pol II present in DE330 (lane 4 to 6); or both 2 ?l of the
anti-Rpb1 flowthrough fraction and 2 ?l of immunopurified Pol II
(lane 7). The relative level of transcription (txn) was quantified by
Phosphorimager, with the maximal level defined as 100 U, and is in-
dicated below each lane.
VOL. 25, 2005TAF-I IS REQUIRED FOR CHROMATIN TRANSCRIPTION 801
tion from chromatin templates may be a general property of
the NAP-1 family of histone chaperones.
TAF-I interactions with chromatin and chromatin remodel-
ing factors. Like other histone chaperones, TAF-I is able to
bind histones in solution (30). Because TAF-I is required for
transcription on chromatin, we asked whether it could interact
with chromatin under transcription conditions. For this exper-
iment, we purified a version of TAF-I? tagged at its amino
terminus with the HA epitope, as well as with His. HA–His–
TAF-I? was as active as His–TAF-I? in the chromatin tran-
scription assay (data not shown). We performed chromatin
immunoprecipitations to determine if TAF-I was interacting
with chromatin during transcription reactions in vitro. Tran-
scription was allowed to proceed for 30 min in the presence of
HA-tagged or untagged TAF-I. The chromatin was then di-
gested with micrococcal nuclease to yield an average fragment
size of 0.5 kb and immunoprecipitated with anti-HA antibody.
We quantified the DNA recovered by PCR with primers flank-
ing the promoter and measured about sixfold enrichment when
the reaction mixture contained HA–His–TAF-I?, relative to
control reaction mixtures containing His–TAF-I? (Fig. 7A).
Thus, TAF-I? can indeed associate with chromatin in the con-
text of transcription.
Because TAF-I is a histone chaperone, one possible expla-
nation for its ability to enhance transcription is that it disrupts or
disassembles chromatin, in effect rendering the plasmid DNA
naked. To test this possibility, different amounts of TAF-I were
incubated with preassembled chromatin prior to limited micro-
coccal nuclease digestion (Fig. 7B). At concentrations equal to or
greater than those that stimulate transcription, TAF-I did not
cause a gross change in chromatin structure detectable by in-
creased accessibility to micrococcal nuclease.
TAF-I belongs to the same family of histone chaperones as
NAP-1, which can participate in chromatin assembly in coop-
eration with the ACF complex. Indeed, TAF-I could substitute
for NAP-1 in the ACF-dependent assembly of chromatin, but
with a 10-fold higher optimal concentration compared to
NAP-1 (Fig. 7C). We also tested whether TAF-I and ACF
physically interact. In pulldown assays with purified proteins,
TAF-I? interacted with the recombinant ISWI-containing
ACF complex (data not shown). SNF2H is the human homolog
of Drosophila ISWI, the catalytic subunit of the ACF complex.
FIG. 4. TAF-I stimulates activated transcription on chromatin templates. (A) Scheme for the further fractionation of the anti-Rpb1
flowthrough fraction. (B, top) VDR-mediated transcription performed in the presence of buffer, 1 ?l of load, or 4 ?l of the indicated Superdex
200 fraction. All reaction mixtures included 0.78 ?g of immunopurified Pol II. (B, bottom) The Superdex 200 fractions were resolved by SDS–12%
PAGE, and proteins were visualized by silver staining. Migration of polypeptides copurifying with the activity—all identified by mass spectrometry
as isoforms of TAF-I—is indicated by asterisks. In, input. The values on the left are molecular sizes in kilodaltons. (C) The active fractions from
the Superdex 200 column were subjected to immunoblotting with a TAF-I antibody that recognizes both the ? and ? isoforms, an ?-specific
antibody, and a ?-specific antibody, as indicated. (D) VDR-mediated transcription performed in the presence of 4 ?l of pooled Superdex 200
fractions 25 and 26 from panel B, buffer only, or 0.14, 0.29, 0.57, 1.14, 2.28, 4.56, 9.12, or 18.24 ?M rTAF-I?. The relative level of transcription
was quantified by Phosphorimager, with the maximal level defined as 100 U, and is indicated below each lane.
802GAMBLE ET AL.MOL. CELL. BIOL.
Incubation of HA–His–TAF-I? with the PC1 fraction, fol-
lowed by anti-HA immunoprecipitation and immunoblotting
with an SNF2H-specific antibody, confirmed an interaction of
SNF2H with TAF-I (Fig. 7D). To our knowledge direct bind-
ing between a NAP family histone chaperone and an ATP-
dependent chromatin-remodeling enzyme has not previously
We explored a possible role of the SNF2H–TAF-I interac-
tion in chromatin transcription by depleting SNF2H from the
PC1 fraction with an anti-SNF2H antibody (Fig. 7E). Com-
pared to mock depletion, depletion of SNF2H had a very small
positive effect on TAF-I-mediated transcription (Fig. 7F, SNF2
depleted). Adding back recombinant ACF to the PC1 fraction
depleted of endogenous SNF2H strongly inhibited transcrip-
tion (Fig. 7F, SNF2 depleted ? rACF). Therefore, SNF2H, a
factor with which TAF-I cooperates to assemble chromatin, is
dispensable (and possibly even inhibitory) for chromatin-tem-
Defining the timing of TAF-I action. Although chromatin
incubated with TAF-I after assembly or chromatin assembled
with TAF-I could not be distinguished from NAP-1-assembled
chromatin by micrococcal nuclease digestion, it remained pos-
sible that TAF-I caused subtle and/or local changes in chro-
matin structure. To test whether TAF-I alone could stably
modify the chromatin to render it transcriptionally competent,
we preincubated rTAF-I? with preassembled chromatin for 30
min prior to S300 spin column chromatography to remove the
TAF-I protein. The pretreated chromatin still required addi-
tion of TAF-I during the transcription reaction, suggesting that
TAF-I was incapable of performing its function in the absence
of other components of the transcription machinery (Fig. 8A).
We also tested the possibility of a functional difference be-
tween chromatin assembled with TAF-I and chromatin assem-
bled with NAP-1 by performing transcription of either tem-
plate with or without additional TAF-I. Without purification of
the templates over S300 spin columns, addition of TAF-I dur-
ing the transcription reaction was only required for the chro-
matin assembled with NAP-1 (Fig. 8B). When the chromatin
was purified to remove factors not stably incorporated during
assembly, however, TAF-I was required during the transcrip-
tion reaction regardless of whether NAP-1 or TAF-I mediated
assembly (Fig. 8B). Therefore, TAF-I facilitates two tempo-
rally distinct processes, assembly of DNA into a chromatin
structure and activation of transcription on a chromatin tem-
Because neither incubation of TAF-I with preassembled
chromatin nor assembly de novo with TAF-I could render
chromatin competent for transcription, TAF-I must act at a
subsequent step to facilitate either preinitiation complex for-
mation, initiation of transcription, or elongation by Pol II
through chromatin. Interestingly, two known Pol II elongation
factors, Spt6 and FACT, are histone chaperones (6, 36). To
test whether TAF-I executes its required function during tran-
script elongation—as does FACT—or at a prior step, we per-
formed pulse-chase transcription on chromatin templates (Fig.
8C). When TAF-I was added prior to a pulse with [?-32P]CTP,
the elongation of labeled transcripts initiated during the pulse
could be observed as a smear extending up the gel after chasing
with an excess of unlabeled CTP. We estimated an elongation
rate of 130 nucleotides/min, comparable to rates previously
measured in vitro on chromatin templates (16). However, if
TAF-I was added only after the reaction mixture had been
supplemented with unlabeled CTP, no elongating transcripts
appeared. Therefore, the requirement for TAF-I is at a step
after the assembly of chromatin but prior to the elongation
phase of transcription.
TAF-I is an activator of chromatin-templated transcription.
We have purified TAF-I from HeLa cell nuclear extracts as a
factor required for chromatin-templated transcription. These
results establish a novel function for NAP family histone chap-
erones in the activation of transcription, which is at odds with
previous reports suggesting a repressive role for TAF-I. There
is recent evidence, however, to support a positive role in vivo.
The Drosophila homolog of TAF-I has been shown to be re-
distributed to heat shock loci, with Pol II, upon transcriptional
activation (35). Therefore, in at least one setting in vivo, TAF-I
is in the right place at the right time to facilitate transcription.
Since its original genetic identification as the product of the
leukemia-associated proto-oncogene SET, TAF-I has had sev-
eral functions ascribed to it. TAF-I was originally purified as a
factor required for the replication in vitro of adenovirus DNA
core particles purified from infected cells. Adenovirus DNA is
FIG. 5. TAF-I is a chromatin-specific coactivator that stimulates
transcription mediated by both VDR and Gal4-VP16. (A) VDR-me-
diated transcription was performed on either naked or chromatin
templates in the presence or absence of 2.28 ?M rTAF-I?, buffer
alone, or 0.024, 0.049, 0.098, 0.195, 0.39, 0.78, or 1.56 ?g of immuno-
purified Pol II, as indicated. (B) Transcription reaction mixtures were
prepared with or without 20 ng of Gal4-VP16 and 3 ?M rTAF-I?, as
indicated, and Gal4-VP16-responsive promoter-containing DNA as-
sembled into chromatin. All reaction mixtures included 0.78 ?g of
immunopurified Pol II. The relative level of transcription was quanti-
fied by Phosphorimager, with the maximal level defined as 100 U, and
is indicated below each lane.
VOL. 25, 2005 TAF-I IS REQUIRED FOR CHROMATIN TRANSCRIPTION 803
packaged into a chromatin-like nucleoprotein structure with
viral basic protein; TAF-I can remodel that structure into a
replication-competent form. Thus, the first and perhaps best-
established role for TAF-I is to promote, rather than impede,
a polymerization reaction on DNA templates assembled into
higher-order chromatin-like structures.
TAF-I was also purified as a potent noncompetitive inhibitor
of protein phosphatase 2A (PP2A), called I2PP2A (25, 26).
Interestingly, PP2A has recently been implicated in mediating
transcriptional repression in Drosophila, leading to the hypoth-
esis that the transcriptional effect of TAF-I might be exerted,
at least in part, through its inhibition of PP2A (35). TAF-I has
also been identified as an inhibitor of the granzyme A-acti-
vated DNase NM23-H1 (11). Granzyme A is inserted into
virus-infected cells by cytotoxic T cells and activates NM23-H1
by cleaving TAF-I, leading to NM23-H1 derepression and in
turn to apoptosis.
Most recent work on TAF-I has emphasized its putative role
as a transcriptional repressor (27, 33, 40, 42). This functional
assignment stems from the ability of TAF-I to inhibit p300-
mediated acetylation of histones in solution. Indeed, TAF-I
was purified from mammalian cell extracts as INHAT (inhib-
itor of histone acetyltransferase) on the basis of this property.
Assays of reporter gene expression after transient transfection
of TAF-I seemed to support a repressive role. These findings
suggest the following three possible (not mutually exclusive)
scenarios: (i) TAF-I is a bona fide corepressor for at least a
subset of transcription factors, (ii) overexpression of TAF-I
represses transcription by promoting the assembly of trans-
fected reporter constructs into generally repressive chromatin
structures, and/or (iii) overexpression of TAF-I above the op-
timal level leads to squelching of transcriptional signals, a
phenomenon we can recapitulate in vitro (Fig. 4D, for exam-
ple). There has been no direct demonstration, however, that
TAF-I represses transcription in vitro or at natural chromo-
somal loci in vivo. Our data, and those obtained in vivo with
Drosophila (35), support a positive role for TAF-I at a subset of
promoters at least. An interesting possibility is that TAF-I can
FIG. 6. Characterization of the TAF-I requirement in chromatin transcription. (A) VDR-mediated transcription performed in the presence of
buffer only or 0.16, 0.33, 0.67, 1.32, or 2.64 ?M rTAF-I? or rTAF-I?, as indicated. (B) Purification of TAF-I?/? heterocomplexes. His-tagged
TAF-I? and FLAG-tagged TAF-I? migrate identically on SDS–10% PAGE, but only TAF-I? can be cleaved by thrombin. Thrombin cleavage of
2.5 ?g of double-affinity-purified TAF-I?/? heterocomplexes or TAF-I? and separation of the products by SDS–10% PAGE reveal a 1:1 ratio of
TAF-I? to TAF-I? in the TAF-I?/? heterocomplex. (C) VDR-mediated transcription performed in the presence of buffer only or 500 nM
His-tagged TAF-I?, His-tagged TAF-I?, FLAG-tagged TAF-I?, or TAF-I?/? heterocomplex. (D, left) VDR-mediated transcription performed in
the presence of buffer only; 0.15, 0.3, 0.6, 1.21, 2.41, 4.82, or 9.65 ?M rTAF-I?(1-225); or 2.28 ?M wild-type rTAF-I? (??). (D, right)
VDR-mediated transcription performed in the presence of buffer only or 0.17, 0.35, 0.69, 1.39, 2.78, 5.56, or 11.11 ?M rTAF-I?(1-255). Wild-type
rTAF-I? (800 nM, ?) was added where indicated. (E) VDR-mediated transcription performed in the presence of buffer only or 0.15, 0.29, 0.58,
1.17, 2.34, 4.67, or 9.35 ?M rTAF-I? or rNAP-1. The relative level of transcription was quantified by Phosphorimager, with the maximal level
defined as 100 U, and is indicated below each lane.
804GAMBLE ET AL.MOL. CELL. BIOL.
be both an activator and a repressor of transcription and that
which effect predominates is context dependent.
In that light, it is perhaps significant that TAF-I has been
purified repeatedly as an inhibitor of one (seemingly unre-
lated) enzyme or another (7, 10, 11, 25, 38, 40). The abilities of
FIG. 7. TAF-I interacts with chromatin and chromatin-remodeling
factors. (A) Transcription reaction mixtures containing HA-tagged or
untagged TAF-I? were incubated for 30 min and then subjected to chro-
matin immunoprecipitation (IP) in vitro with HA antibody. Coimmuno-
gel electrophoresis. (B) Preassembled chromatin was incubated with
buffer alone (?) or 15.3 (?) or 53 (??) ng of HeLa TAF-I from the peak
Superdex 200 fractions per ?l (Fig. 4B) and then subjected to limited
micrococcal nuclease (MNase) digestion. A 15.3-ng/?l concentration of
this fraction is roughly equivalent to 4 ?l of fractions 25 and 26 assayed in
Fig. 4B. (C) Chromatin assembly reaction mixtures were prepared with-
out rNAP-1. Where indicated, ACF and 8 ?M rTAF-I? were present
during assembly. After assembly, the chromatin was subjected to limited
micrococcal nuclease digestion. (D) HA-tagged or untagged TAF-I? (58
pmol) was incubated with 36 ?g of the PC1 fraction for 30 min and
subsequently immunoprecipitated with HA antibody. The immunopre-
cipitates and input (10%) were analyzed by immunoblot assay with an
anti-SNF2 antibody. (E) The mock-depleted or anti-SNF2-depleted PC1
fraction was analyzed by immunoblotting with the anti-SNF2 antibody.
(F) Transcription reaction mixtures were prepared with 3 ?g of PC1,
mock-depleted PC1, or anti-SNF2-depleted PC1 on unpurified and S300
spin column-purified chromatin (chr). rACF (170 nM) and rTAF-I? (3
?M) were added where indicated. The relative level of transcription was
quantified by Phosphorimager, with the maximal level defined as 100 U,
and is indicated below each lane.
FIG. 8. TAF-I is required at a step subsequent to chromatin as-
sembly and prior to transcript elongation. (A) NAP-1-assembled chro-
matin was preincubated with or without 7.5 ?M rTAF-I? for 30 min as
indicated. The chromatin was used directly or passed over an S300 spin
column. Transcription reaction mixtures were prepared with (?) or
without (?) 3 ?M rTAF-I?. (B) Chromatin (Chr) assembled with 0.9
?M NAP-1 or 8 ?M rTAF-I? was used directly or purified over an
S300 spin column, as indicated. Transcription reaction mixtures were
then prepared with (?) or without (?) 3 ?M rTAF-I?. The relative
level of transcription in panels A and B was quantified by Phosphor-
imager, with the maximal level defined as 100 U, and is indicated below
each lane. (C) Transcription elongation assays were performed with
buffer alone or with 4 ?M TAF-I? added before the pulse (TAF-I?) or
after the chase (TAF-I? postchase), as indicated. Samples were taken
after the pulse (P) or at 0.5, 1, 1.5, 3, 6, or 18 min of chase and analyzed
on a 6% denaturing gel. The smear of radioactivity extending up the
gel indicates elongation of transcripts initiated during the pulse and
was only seen when ??F-I? was included prior to the chase. The
values on the left are sizes in nucleotides.
VOL. 25, 2005 TAF-I IS REQUIRED FOR CHROMATIN TRANSCRIPTION 805
TAF-I to inhibit several different enzymes and interact stably
with a multitude of proteins in extracts (unpublished observa-
tions) may be functions of its highly acidic carboxyl-terminal
region. Whatever the explanation, given the complexity of the
protein-protein interactions possible for TAF-I, the true func-
tion(s) of this protein may be difficult to discern by considering
any one interaction in isolation. We have taken a different
approach to show that TAF-I plays a positive, required role in
a complex biochemical process reconstituted in vitro: tran-
scription from promoter DNA embedded in chromatin.
Requirement of a histone chaperone early in the transcrip-
tion cycle. We have shown that TAF-I not only stimulates
activator-mediated transcription on chromatin but also can
cooperate with ACF in the assembly of chromatin. A role for
histone chaperones in the assembly of chromatin is experimen-
tally well supported (2, 28). Histone chaperones such as CAF-I
and Asf1 participate in replication-coupled pathways, whereas
others, such as NAP-1, participate with ISWI family members
in replication-independent chromatin assembly.
Histone chaperones have also recently been implicated di-
rectly in transcriptional activation as components of a tran-
scription-coupled chromatin assembly or maintenance path-
way. In vivo, regions of DNA actively transcribed by Pol II
retain a chromatin structure. At a physiologic salt concentra-
tion, however, even a single nucleosome is a strong barrier to
Pol II transcription (19). In the presence of either a high salt
concentration or the chromatin-specific transcription elonga-
tion factor and histone chaperone FACT, Pol II transcription
disrupts chromatin structure by displacement of an H2A-H2B
dimer from the nucleosome (4, 19). Mutation of SPT16 (which
encodes a component of FACT) in yeast leads to disruption of
chromatin structure specifically in actively transcribed regions,
leading to aberrant initiation from cryptic start sites and mis-
regulation of certain transcripts (18). We attempted to discern
a similar effect of TAF-I on elongation, but because of its
required role at an earlier step, any effect of TAF-I analogous
to that of FACT could not be measured.
We have identified another step in the transcription cycle at
which a histone chaperone is required to relieve chromatin-
mediated repression: after chromatin assembly and prior to the
elongation phase. TAF-I may be needed, for example, to
remodel chromatin into a state competent for preinitiation
complex assembly. Indeed, recent evidence from two groups
supports a role for histone chaperones in remodeling the chro-
matin of promoters to activate transcription in vivo. At the
yeast PHO5 and PHO8 promoters, the chromatin conforma-
tional changes that occur upon activation of transcription are
due to nucleosome disassembly rather than the sliding of nu-
cleosomes mediated by remodeling enzymes (5). Furthermore,
the histone chaperone Asf1 is required both for the creation of
the nuclease-hypersensitive chromatin structure at these pro-
moters and for their transcriptional activation (1).
Model for TAF-I function in transcriptional activation.
Might TAF-I act in a pathway analogous to that of Asf1 to
disassemble chromatin at a promoter destined for transcrip-
tional activation? Support for a role in chromatin disassembly
comes from TAF-I’s ability to bind and trigger the deconden-
sation of sperm chromatin, with simultaneous release of
sperm-specific, histone-like basic proteins from the chromatin
(29, 30). Furthermore, two independent reports have demon-
strated that the related (and in our assays, functionally equiv-
alent) NAP-1 protein can mediate activator-dependent disrup-
tion of nucleosomes (14, 44). Our experiments indicate,
however, that incubation of TAF-I by itself with chromatin is
insufficient to render the chromatin transcriptionally compe-
tent (Fig. 8). Therefore, TAF-I is likely to act in concert with
other transcription factors or chromatin-remodeling activities
to activate transcription. In summary, we have uncovered a
positive role for TAF-I and its relatives in transcribing chro-
matin and have defined a window for that action early in the
transcription cycle. We may now ask whether this is a general
requirement in vivo and how these conserved histone chaper-
ones activate transcription on chromatin.
We thank Stewart Shuman and Ste ´phane Larochelle for critical
review of the manuscript. We are grateful to James Kadonaga, who
provided the NAP-1, ISWI, and Acf1 baculoviruses; Kyosuke Nagata,
who provided the anti-TAF-I antibodies; Jeffrey Parvin, who provided
the TFIIA expression vector; and W. Lee Kraus, who provided the
Gal4-VP16 expression vector and the pGEIO plasmid. HeLa cells
were cultured by the National Cell Culture Center. We thank Brian
Lemon for helpful discussion and Chao-Pei Chang for experimental
This work was supported by NIH grant DK45460 to L.P.F. and
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