THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 280, No. 30, Issue of July 29, pp. 27523–27532, 2005
Printed in U.S.A.

High Mobility Group Protein-B1 Interacts with Sterol Regulatory
Element-binding Proteins to Enhance Their DNA Binding*Received for publication, December 27, 2004, and in revised form, April 21, 2005
Published, JBC Papers in Press, May 12, 2005, DOI 10.1074/jbc.M414549200§

Yuho Najima‡, Naoya Yahagi‡, Yoshinori Takeuchi§, Takashi Matsuzaka§, Motohiro Sekiya‡,
Yoshimi Nakagawa§, Michiyo Amemiya-Kudo‡, Hiroaki Okazaki‡, Sachiko Okazaki‡,
Yoshiaki Tamura‡, Yoko Iizuka‡, Ken Ohashi‡, Kenji Harada‡, Takanari Gotoda‡, Ryozo Nagai‡,Takashi Kadowaki‡, Shun Ishibashi‡, Nobuhiro Yamada§, Jun-ichi Osuga‡,and Hitoshi Shimano§¶
From the ‡Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655 and the§Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki 305-8575, Japan

Sterol regulatory element-binding proteins (SREBPs)
are transcription factors that are predominately involvedin the regulation of lipogenic and cholesterogenic enzyme
gene expression. To identify unknown proteins that interact with SREBP, we screened nuclear extract proteins
with 35S-labeled SREBP-1 bait in Far Western blotting
analysis. Using this approach, high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified
as a novel SREBP interacting protein. In vitro glutathione
S-transferase pull-down and in vivo coimmunoprecipitation studies confirmed an interaction between HMGB1
and both SREBP-1 and -2. The protein-protein interaction
was mediated through the helix-loop-helix domain of
SREBP-1, residues 309 –344, and the A box of HMGB1.
Furthermore, an electrophoretic mobility shift assay
demonstrated that HMGB1 enhances SREBPs binding totheir cognate DNA sequences. Moreover, luciferase reporter analyses, including RNA interference technique
showed that HMGB1 potentiates the transcriptional activities of SREBP in cultured cells. These findings raise
the intriguing possibility that HMGB1 is potentially involved in the regulation of lipogenic and cholesterogenic
gene transcription.

Sterol regulatory element-binding proteins (SREBPs)1 aremembers of the basic helix-loop-helix leucine zipper (B-HLHZIP) family of transcription factors that bind to specific sterolresponsive elements (SREs) in the promoters of target genes
and thereby regulate fatty acid and cholesterol synthesis (reviewed in Refs. 1–3). Two homologous genes, Srebp-1 andSrebp-2, have been identified. The Srebp-1 gene is furthertranscribed into two isoforms, SREBP-1c and -1a, by distinct
promoters. In lipogenic organs such as liver and adipose tis* This work was supported by a grant from the Research Fellowshipsof the Japan Society for the Promotion of Science (to N. Y.). The costs ofpublication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-29-8632081; Fax: 81-29-853-3174; E-mail: shimano-tky@umin.ac.jp.
1
The abbreviations used are: SREBP, sterol regulatory elementbinding protein; B-HLH-ZIP, basic helix-loop-helix leucine zipper; SRE,
sterol-responsive element; HMG, high mobility group; HMGB1, highmobility group protein-B1; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; TBS, Tris-buffered saline; GST, glutathione S-transferase; EMSA, electrophoretic
mobility shift assay; PSD, post-source decay; TR-FRET, time-resolved
fluorescence resonance energy transfer.
This paper is available on line at http://www.jbc.org

sues, the predominant isoform is SREBP-1c, which controls
gene expression of lipogenic enzymes, including fatty acid synthase and ATP citrate lyase, whereas SREBP-2 plays a crucialrole in the regulation of key enzymes in the cholesterol synthetic pathway such as 3-hydroxy-3-methylglutaryl (HMG)CoA synthase and farnesyl diphosphate synthase (4 –9). However, the molecular mechanisms that determine this selectivity
have not been entirely elucidated in the context of their preference for DNA sequence (10). It is likely related to theircomplicated interactions with various transcription factors andcofactors (11).
Several nuclear proteins that act as modulators of SREBPregulated transcription have been identified. Some of them
function as activators (e.g. CREB-binding protein (12), Sp1(13), and NF-Y (14)) and some as repressors (e.g. YY1 (15, 16),
ATF6 (17), and WT1 (18)). Recently, we and others reported
that hepatocyte nuclear factor 4 is another interacting factor(19, 20). Taken together, it has been demonstrated that
SREBPs, like other transcription factors, interact with a broad
range of proteins to control various signaling pathways. To
better understand the precise mechanisms of SREBP regulation, a comprehensive study to identify unknown interacting
factors is imperative.
In the present study, we set out to identify these proteins byscreening nuclear extract proteins using Far Western blotting
technique and identified high mobility group protein-B1(HMGB1) as a new factor interacting with SREBP by matrixassisted laser-desorption ionization and time-of-flight (MALDITOF) mass spectrometry. HMGB1 is a chromatin-binding protein that belongs to high mobility group (HMG)-box family andappears to act as an architectural facilitator in the assembly of
nucleoprotein complexes in a variety of DNA-related processes
including transcription, replication, V(D)J recombination, andrepair (reviewed in Refs. 21 and 22). Here, we describe thefurther function of HMGB1 in interacting with SREBPs.
MATERIALS AND METHODS

Nuclear Protein Extraction
Nuclear extracts from mice livers were prepared as previously described (23). Briefly, excised livers (0.5 g) from 24-h fasted or 12-h refed
C57BL/6J mice were homogenized in a Polytron in 5 ml of buffer A (10mM Hepes at pH7.9, 25 mM KCl, 1 mM EDTA, 2 M sucrose, 10% glycerol,
0.15 mM spermine, and 2 mM spermidine, supplemented with proteaseinhibitors (12.5 ␮g/ml N-acetyl-Leu-Leu-norleucinal (Calbiochem), 2.5
␮g/ml pepstatin A, 2 ␮g/ml leupeptin, 0.1 mM phenylmethylsulfonyl
fluoride, and 2.5 ␮g/ml aprotinin). Pooled homogenate was then subjected to one stroke of a Teflon pestle in a Potter-Elvehjem homogenizer,
followed by filtration through two layers of cheesecloth, and layered

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HMGB1 Activates SREBP

over 10 ml of buffer A in a Beckman SW28 rotor. After centrifugation at
24,000 rpm for 1 h at 4 °C, the resulting nuclear pellet was resuspended
in a buffer containing 10 mM Hepes at pH 7.9, 100 mM KCl, 2 mM MgCl2,
1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol supplemented withprotease inhibitors, after which 0.1 volume of 5 M NaCl was added. Eachmixture was agitated gently for 30 min at 4 °C and then centrifuged at
89,000 rpm in a Himac S120AT2 rotor (Hitachi, Tokyo, Japan) for 30
min at 4 °C. The supernatant was used as nuclear extract.

Coimmunoprecipitation
Aliquots of nuclear extract protein (100 ␮g) were diluted in binding
buffer (20 mM Hepes at pH 7.9, 150 mM NaCl, 1 mM EDTA, 1% Triton
X-100, 0.1 mM phenylmethylsulfonyl fluoride, 12.5 ␮g/ml N-acetyl-LeuLeu-norleucinal, and 2 ␮g/ml leupeptin as final concentration), andwere subjected to immunoprecipitation using a rabbit polyclonal or amouse monoclonal anti-SREBP-1 antibody (24, 25) and protein
G-Sepharose beads (Amersham Biosciences). After 6-h incubation at
4 °C, the beads were washed four times in wash buffer (20 mM Hepes at
pH 7.9, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 supplemented
with protease inhibitors) and were then boiled briefly in SDS-PAGE
sample buffer to elute proteins for subsequent electrophoresis.

Western Blotting
Following SDS-PAGE, immunoprecipitated proteins were transferred onto nitrocellulose membrane (Hybond ECL, Amersham Biosciences). SREBP-1 and HMGB1 were detected using a 1:1000 dilutionof rabbit polyclonal anti-SREBP-1 and anti-HMGB1 (BD Pharmingen)antibodies, respectively, in TBS buffer (20 mM Tris-HCl at pH 7.6 and140 mM NaCl) containing 0.2% Tween 20 (Bio-Rad) and 5% skimmed
milk. Bound antibodies were detected with an horseradish peroxidasecoupled anti-rabbit IgG secondary antibody (Amersham Biosciences)and visualized using ECL chemiluminescent substrates (Amersham
Biosciences). For Western blot experiments, immunoprecipitation was
performed using mouse monoclonal antibody to avoid background signals from immunoprecipitation antibody detected by secondary
antibody.

Far Western Blotting
Nuclear extract or immunoprecipitated protein was blotted onto
polyvinylidene difluoride membrane (Hybond P, Amersham Biosciences), was incubated for 1 h at room temperature in blocking buffer(TBS buffer containing 0.2% Tween 20 and 5% skimmed milk) and wasthen mixed overnight at 4 °C with 35S-labeled protein bait in the same
buffer. [35S]Methionine-labeled SREBP-1c bait was generated by using
an in vitro transcription/translation system using reticulocyte lysate(TNT T7 quick-coupled transcription/translation system, Promega), andwas directly added to the buffer. After washing three times with TBS
buffer containing 0.2% Triton X-100, signals were visualized with
BAS2000 (Fuji photo film, Tokyo, Japan).

Protein Identification by MALDI-TOF MS
Peptide Mass Fingerprinting—Proteins were separated by SDSPAGE and stained with Coomassie Brilliant Blue. The visualized band
was excised, destained, washed four times in wash buffer (50% acetonitrile and 25 mM NH4HCO3 at pH 8.0), reduced in reducing buffer (100mM dithiothreitol and 100 mM NH4HCO3) at 56 °C for 30 min, andalkylated with 100 mM acrylamide (in 100 mM NH4HCO3) at 37 °C for
30 min. The gel slice was then dehydrated with acetonitrile (highperformance liquid chromatography-grade, Wako Chemicals) and rehydrated with 10 ␮l of 50 mM NH4HCO3 (pH 8.0) containing 10 ␮g/␮lmodified trypsin (sequencing grade, Promega), and trypsin in-gel digestion was carried out for 8 h at 37 °C. The resulting peptides wereextracted with 66% acetonitrile-0.1% trifluoroacetic acid (high-performance liquid chromatography-grade, Wako Chemicals), condensed by
vacuum centrifugation, and desalted by ZipTipC18 pipette tips (Millipore). Following elution into 2 ␮l of 60% acetnitrile-0.1% trifluoroacetic
acid, 0.5 ␮l was applied to a target plate, overlaid with 0.5 ␮l of␣-cyano-4-hydroxycinnamic acid (Sigma) matrix, and evaporated atroom temperature. Matrix-assisted laser-desorption ionization andtime-of-flight (MALDI-TOF) mass spectrometry was performed using a
mass spectrometer (Voyager-DE STR, Applied Biosystems) operated indelayed extraction and reflectron mode. The obtained mass spectrum
was calibrated externally using standard peptides (des-ARG-bradykinin, [Glu-1]fibrinopeptide B, and adrenocorticotropic hormone (clip
1–17): m/z ⫽ 904.4681, 1570.6774, and 2093.0867, respectively, PE
biosystems) and was then calibrated internally within 20 ppm using
trypsin autolytic peptides present in the sample (m/z ⫽ 842.5100,

1045.5642, and 2211.1046). Obtained data of monoisotopic ion mass([M⫹H]) were entered into the National Center for Biotechnology Information (NCBI) data base, searching for corresponding protein by
MS-Fit algorithm (prospector.ucsf.edu).
Post-source Decay Analysis—To confirm the protein identification,
PSD spectra were acquired at delayed extraction and reflectron mode.
The accelerating voltage was set at 20 kV, grid voltage at 75%, guidewire voltage at 0.024%, and delay time at 100 ns. The timed ion selector
was pre-set to the [M⫹H] mass of the peptides. The spectra wereacquired in 10 –13 segments with mirror ratios 1.0 – 0.13 and assembled
by using instrument software (DataExproler). Obtained peptide masses
were used to search the NCBI data base by using the MS-Tag algorithm(prospector.ucsf.edu).

Plasmid Constructions
A series of truncated derivatives of mouse SREBP-1c cDNA expression plasmids used for in vitro transcription/translation in GST pulldown assays were constructed by inserting each fragment into
pcDNA3.1(⫹) expression vector (Invitrogen) as follows: SREBP-1-FL(amino acid residues 1– 453) cDNA fragment was cut out of pTKSREBP-1c construct described previously (26) by NotI and XbaI followed by insertion into pcDNA3.1(⫹) at NotI and XbaI sites; SREBP1-B-HLH-ZIP (residues 1–364) and SREBP-1-B-HLH (residues 1–344)
were constructed by amplifying each segment by PCR with a 5⬘ primer,
including NheI site and initiation codon (ATG), and 3⬘ primers, including a stop codon (TAA) and SalI site, and by inserting it into NheI and
XhoI sites of pcDNA3.1(⫹); SREBP-1-Basic (residues 1–309) was constructed by inserting NotI-AgeI fragment attached with an adapter,
including Age I site and a stop codon into pcDNA3.1(⫹) at NotI and
XbaI sites; SREBP-1-⌬(B-HLH-ZIP) (residues 1–285) was constructedby ligating NotI-AvrII (AvrII site was blunted with Klenow DNA polymerase) fragment into pcDNA3.1(⫹) at NotI and XbaI (also blunted)
sites; SREBP-1-C-terminal (residues 365– 449) was PCR-amplified with5⬘-primer, including an initiation codon and inserted into EcoRI and
XhoI sites of pcDNA3.1(⫹). SREBP-1-⌬TA (residues 91– 460) was as
described previously (20). Transcription from these expression plasmids
was driven by T7 promoter when used in TNT T7 quick-coupled transcription/translation system (Promega).
A prokaryotic expression plasmid with His6 tag at N-terminal used
for generating SREBP-1c recombinant protein was constructed as follows: full-length SREBP-1c cDNA fragment cut out of pTK-SREBP-1cconstruct described previously (26) by NotI and XbaI and blunted atboth ends with Klenow DNA polymerase was inserted into NotI
(blunted) and SmaI sites of pIVEX2.4d (Roche Applied Science), where
NotI sites were blunted in order for the frame not to be shifted. To
construct an expression plasmid for GST-SREBP-1 fusion protein,
cDNA corresponding to amino acid residues 286 –364 (basic helix-loophelix leucine zipper region) was PCR-amplified and inserted into pGEX4T2 (Amersham Biosciences) at BamHI and SalI sites without frameshift. A full-length SREBP-2 cDNA construct in pcDNA3.1(⫹) was
described previously (20).
To construct HMGB1 expression plasmids, mouse full-length
HMGB1 cDNA reverse-transcribed from liver total RNA was amplified
by PCR and cloned into pGEM-T-easy vector (Promega). After sequencing the plasmid, full-length cDNA fragment cut out by EcoRI andblunted at both ends was inserted into XhoI sites (blunted) of
pIVEX2.4d to construct His-HMGB1-FL. For His-HMGB1-Abox (aminoacid residues 1–97), EcoRI (cut and blunted), StuI fragment was cloned
into XhoI site (blunted) of pIVEX2.4d. Similarly, His-HMGB1-⌬Abox(amino acid residues 98 –215) was constructed by inserting StuI-EcoRI(blunted) fragment into NcoI site (blunted) of pIVEX2.4d. Those plasmids were used for generating recombinant proteins both in BL21(DE3)
Escherichia coli and in in vitro transcription/translation system (Rapid
Translation System, Roche Applied Science). To construct an expression plasmid for GST-HMGB1 fusion protein, EcoRI fragment wasinserted into pGEX-4T2 (Amersham Biosciences) at the EcoRI site. All
the constructs were verified by sequencing.

Generation of Recombinant Proteins[35S]Methionine-labeled proteins were generated from pcDNA3.1(⫹)
expression plasmids (Invitrogen) using T7 RNA polymerase and reticulocyte lysate (TNT T7 quick-coupled transcription/translation system,
Promega) or from pIVEX2.4d plasmids utilizing E. coli lysate with T7
RNA polymerase (Rapid Translation System, Roche Applied Science)
according to the manufacturer’s protocol. GST and GST fusion proteins
were expressed in E. coli (DH5␣) using pGEX-4T (Amersham Biosciences) and purified using glutathione-Sepharose beads (Amersham

HMGB1 Activates SREBP

27525

Biosciences) by standard method. His-tagged proteins were expressedwith constructs using pIVEX2.4d in BL21(DE3) strain of E. coli andpurified by binding to Ni-IDA resin (ProBond Purification System,
Invitrogen) and eluting with imidazole followed by dialysis. Full-length
HMGB1 protein without His tag was obtained with protease cleavage
followed by treatment with benzamidine-Sepharose (Amersham
Biosciences).

GST Pull-down Assays
GST pull-down assays were performed according to standard protocol. Briefly, GST or GST fusion proteins attached onto glutathioneSepharose beads (Amersham Biosciences) in advance were mixed withan [35S]methionine-labeled target proteins in binding buffer (20 mM
Hepes at pH 7.9, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with protease inhibitors). After 2 h of incubation at 4 °C, thebeads were washed four times in wash buffer (20 mM Hepes at pH 7.9,
150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 supplemented withprotease inhibitors), and were boiled briefly in SDS-PAGE sample
buffer to elute proteins for subsequent electrophoresis. Gels were dried,and signals were visualized with BAS2000 imaging system (Fuji photofilm).

Electrophoretic Mobility Shift Assays
Electrophoretic mobility shift assays were performed as previously
described (27). Briefly, the DNA probe was prepared by annealing two
oligonucleotides 5⬘-GAGATCACCCCACTGCAAACTCCTCCC-3⬘ and
5⬘-GCAGGGGGAGGAGTTTGCAGTGGGGTG-3⬘, which correspond toboth strands of the sterol regulatory element (SRE) in the promoter oflow density lipoprotein receptor gene (28), and labeling them with
[␣-32P]dCTP by filling in the 5⬘-overhangs with Klenow DNA polymerase (Amersham Biosciences), followed by purification on Sephadex G-50
(Amersham Biosciences) columns. The labeled DNA probe was incubated with a recombinant His-SREBP-1 or -2 protein (200 ng) mixed
with various amount of GST-HMGB1 or GST alone in a buffer containing 10 mM Hepes at pH 7.8, 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 5 mM
dithiothreitol, 30 ␮g/ml poly(dI-dC), and 0.1% Triton X-100, for 30 min
on ice. The DNA䡠protein complexes were resolved on 4.6% polyacrylamide gels in 1⫻ TBE buffer or glycine buffer (50 mM Tris-HCl at pH
8.0, 380 mM glycine, and 2 mM EDTA). Supershift reactions were performed by adding anti-SREBP-1 or -2 polyclonal antibody.

Preparation of Short Hairpin RNA Expression Cassettefor RNAi Analysis
The iGENE algorithm (iGENE Therapeutics) was used to list candidates of RNA interference target sequences for human HMGB1. For six
candidates short hairpin RNA expression plasmids were constructed bycloning annealed oligonucleotides into pENTR/U6 expression vector(BLOCK-iT U6 RNAi Entry Vector Kit, Invitrogen). The most effective
construct was selected through transfection experiments (data notshown). The selected target sequence was 5⬘-AGACCTGAGAATGTATCCCCAAA-3⬘ on the 3⬘ non-coding region and annealed oligonucleotides were 5⬘-CACCACTTGAGAGTGTATCTCCAACGTGTGCTGTCCGTTGGGGATACATTCTCAGGT-3⬘ and 5⬘-AAAAACCTGAGAATGTATCCCCAACGGACAGCACACGTTGGAGATACACTCTCAAGT-3⬘.

Transfection and Luciferase Assays
Luciferase reporter plasmid with sterol regulatory element (pSRELuc) was previously described (7). Human embryonic kidney 293 cells
were grown in Dulbecco’s modified Eagle’s medium containing 10%
fetal bovine serum, penicillin, and streptomycin. Cells were transfectedwith pSRE-Luc and the indicated expression plasmids using SuperFect
Transfection Reagent (Qiagen). After a 24-h incubation, the luciferase
activity in transfectants was measured on a luminometer by using astandard assay kit (Promega).

Measurement of Time-resolved Fluorescence
Resonance Energy Transfer
For detecting interaction between SREBP-1 and HMGB1, a TRFRET assay was performed by incubating 10 nM GST-SREBP-1 or GST
alone, and 10 nM allophycocyanin-conjugated anti-GST antibody (SureLight, PerkinElmer Life Sciences) with 0 –100 nM His-HMGB1-FL,
His-HMGB1-Abox, or His-HMGB1-⌬Abox recombinant protein and 1
nM europium-conjugated anti-His6 antibody (Lance, PerkinElmer Life
Sciences) in FRET assay buffer (10 mM Tris-HCl at pH 8.0, 150 mM
NaCl, and 0.1% bovine serum albumin, supplemented with proteaseinhibitors) for 8 h at 4 °C. Time-resolved fluorescence exited at 340 nm
and detected at 665 nm with delay time of 50 ␮s was measured by

FIG. 1. Far Western blotting analysis detecting hepatic nuclear proteins associated with SREBP-1c. a, Far Western analysisof mice hepatic nuclear extracts using 35S-labeled SREBP-1c as a bait.
Aliquots of nuclear proteins (25 ␮g) extracted from fasted (F) and refed(R) mice livers were subjected to SDS-PAGE. [35S]Methionine-labeled
SREBP-1c bait was generated by in vitro transcription/translation system. b, immunoprecipitation of SREBP-1 from nuclear proteins (100 ␮g)with anti-SREBP-1 antibody. Immunoprecipitates with a mouse monoclonal antibody were subjected to immunoblotting with a rabbit polyclonal antibody. c, Far Western analysis of SREBP-1-coimmunoprecitated proteins. The same proteins as in b were blotted and incubatedwith [35S]methionine-labeled SREBP-1c. SREBP-1 was detected by its
homodimerization.
cumulative counts of 1000 repeats on an ARVO MX analyzer(PerkinElmer Life Sciences). The results are expressed as 1000 ⫻ (665
nm/615 nm). For experiments examining SREBP-1 dimerization, similar TR-FRET assays between GST-SREBP-1 and His-SREBP-1 (10 nMeach) with or without 1 nM no-tagged-HMGB1 were performed.
RESULTSFar Western Blot Demonstrating That SREBP-1 Binds to
Several Nuclear Proteins in the Liver—In an attempt to search
for new interacting factors with SREBP, we first performed Far
Western blotting analysis of nuclear proteins extracted frommouse livers. As shown in Fig. 1a, several bands were detectedby 35S-labeled SREBP-1c (nuclear form) bait. To further examine whether these proteins are coimmunoprecipitated with
SREBP-1 using anti-SREBP-1 antibody, immunoprecipitatednuclear proteins were analyzed by Far Western technique inthe same way. Because hepatic SREBP-1 is scarce when animals are fasted and is markedly induced when animals arerefed after starvation as reported previously (29) and shown in
Fig. 1b, we compared nuclear proteins from refed mouse livers
with those from fasted animals as a negative control. As shownin Fig. 1c, 35S-labeled SREBP-1c bait detected three bands
around 30 –35 kDa only in refed mice, suggesting that these
nuclear proteins physiologically interact with SREBP-1c. Homodimerization of SREBP-1 was also detected at 66 kDa,
which demonstrates that this Far Western blotting analysis isequally sensitive and specific with Western blotting.
Identification of the Protein as HMGB1 Using MALDI-TOF
MS—Next we proceeded to identification of these nuclear proteins. The three strong bands were noticed by Ponceau-stainingof crude nuclear bands exactly at the same positions as by Far
Western analysis. Therefore, total nuclear proteins were resolved by SDS-PAGE, stained with Coomassie Brilliant Blue
(Fig. 2a). The three corresponding bands were excised, applied
to in-gel trypsin digestion and analyzed by peptide mass fingerprinting method using MALDI-TOF MS.
Fig. 2b shows a representative MALDI-TOF MS spectrum
obtained from a trypsin-digested sample of the lowest band(around 30 kDa). These dozens of peaks were analyzed with
MS-fit (prospector.ucsf.edu) peptide mass fingerprinting algorithm, and a protein named high mobility group protein-B1

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HMGB1 Activates SREBP

FIG. 2. Protein identification by MALDI-TOF MS. a, Coomassie Brilliant Blue (CBB) staining of hepatic nuclear proteins resolved by
SDS-PAGE. The band indicated by the arrowhead was excised and subjected to trypsin digestion followed by peptide mass analyses. b, a
representative spectrum of trypsin-digested peptides obtained on a MALDI-TOF MS analyzer. Peaks denoted by the asterisk were identified asoriginating from HMGB1 by standard peptide mass fingerprinting (PMF) algorithm (MS-Fit). c, spectra resulted from post-source decay (PSD)
analysis. Three parent ions denoted by the # symbol in b were further analyzed in PSD mode, and a representative composite mass spectrum offragment ions from a precursor of 1510.7 Da, denoted by #2 in b, is displayed. Series of b and y ions (b4 – b9 and y7–y10) were detected, and a partial
amino acid sequence (MSSYAFFVQTCR, corresponding to HMGB1 amino acid residues 13–24) was determined. Similarly, precursors denoted by#1 and #3 were revealed to be HMGB1 amino acid residues 155–163 and 129 –146, respectively.

(HMGB1) was identified with by far the highest probability.
The other two bands were also identified as other proteins.
To reinforce this identification, post-source decay (PSD)
analyses of amino acid sequence were also performed. Threeparent ions denoted by #1 through #3 in Fig. 2b were further
analyzed in PSD mode, and spectra were assembled by the
instrument software (Fig. 2c). By searching the NCBI database with the MS-Tag algorithm (prospector.ucsf.edu), thethree precursor ions were all identified as HMGB1.
In Vivo Coimmunoprecipitation of HMGB1 by Anti-SREBP-1
Antibody—To validate this identification of the band observed
by Far Western as HMGB1, the in vivo coimmunoprecipitatednuclear proteins by anti-SREBP-1 antibody were immunoblotted using anti-HMGB1 antibody (Fig. 3). To avoid background signals from secondary antibody detecting immunoprecipitation antibody, immunoprecipitation was performed withmouse monoclonal antibody and immunoblotting was with rabbit polyclonal antibody. Because fasted mouse livers containbarely detectable levels of SREBP-1, they were used as a negative control. As expected, the HMGB1 band was detected only
in refed mice where SREBP-1 existed abundantly and thereby
coimmunoprecipitated HMGB1, whereas fasted mice liver exhibited no HMGB1 bands. These results demonstrate that
SREBP-1 binds to HMGB1in vivo in the nuclei of hepatocytes.
SREBP-1 Interacts with HMGB1 through HLH Domain—We
further investigated the binding of SREBP-1 to HMGB1 by GST
pull-down assay. As expected, the full-length nuclear form of
SREBP-1c (amino acid residues 1– 453) generated and labeledwith [35S]methionine by in vitro transcription/translation systemwere pulled down by recombinant GST-HMGB1 fusion protein(Fig. 4c). In contrast, GST control resin showed no interaction.

Next we attempted to determine the domain of SREBP-1
responsible for the interaction with HMGB1; SREBP-1 consistsof several domains such as acidic, basic (B), helix-loop-helix(HLH), and leucine zipper (ZIP) domains (Fig. 4a). For thispurpose, deletion derivatives of SREBP-1c were generated andlabeled with [35S]methionine using the in vitro transcription/translation system and were subjected to GST pull-down assay
with GST-HMGB1 (Fig. 4, d and e). In the series of deletion
derivatives sequentially deleted from the C-terminal,
SREBP-1c with amino acid residues 1–344 or longer (with HLH
domain (309 –344)) were pulled down by GST-HMGB1,
whereas those with amino acid residues 1–309 or shorter (without HLH domain) were not. These findings demonstrate that
HLH domain of SREBP-1 is critical for its interaction with
HMGB1.
SREBP-1 and -2 are homologous with each other, and inparticular, their HLH domains have the highest similarity of
77% in their amino acid sequence (Fig. 4f). Therefore, we examined whether SREBP-2 also binds to HMGB1 in the samepull-down assay. As expected, SREBP-2 was pulled down with
GST-HMGB1 (Fig. 4g), demonstrating that SREBP-2 as well as
SREBP-1 binds to HMGB1.
HMGB1 Interacts with SREBP-1 through its A Box—To precisely understand the interaction between SREBP-1 andHMGB1 and at the same time to map the site of HMGB1
required for this interaction, we performed another GST pulldown assay in the reverse setting using a GST-SREBP-1 fusion
protein containing B-HLH-ZIP domain of SREBP-1 (Fig. 5b),and recombinant proteins with deleted domains of HMGB1.
HMGB1 is structured into two HMG box domains (A and B
boxes, 30% identical in amino acid sequence) plus a highly

HMGB1 Activates SREBP

FIG. 2— continued

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HMGB1 Activates SREBP

acidic C-terminal region (Fig. 5a). We therefore generated thefollowing three 35S-labeled recombinant proteins with in vitrotranscription/translation system: full-length HMGB1 protein,
HMGB1-Abox containing A box alone, and HMGB1-⌬Abox
lacking A box and containing B box and C-terminal. To avoid
carryover of HMGB1 protein included in the reaction reagent
from reticulocytes, we used an E. coli lysate system. As expected, HMGB1 exhibited specific binding to B-HLH-ZIP domain of SREBP-1 (Fig. 5c). It was further revealed that the Abox of HMGB1 was sufficient for this binding, whereas protein
without A box (⌬Abox) was not capable of binding (Fig. 5c).
These data demonstrate that the A box of HMGB1 and the

FIG. 3. In vivo coimmunoprecipitation of HMGB1 with
SREBP-1. Coimmunoprecipitation of HMGB1 with SREBP-1 using
anti-SREBP-1 antibody was shown. Anti-SREBP-1-immunoprecipitates from hepatic nuclear proteins (100 ␮g) was resolved by SDSPAGE followed by Western blotting analysis with an anti-HMGB1
antibody. Nuclear extract proteins were prepared from livers of mice ina fasted (F) and refed (R) state.

B-HLH-ZIP domain of SREBP-1 have a direct interaction.
HMGB1 Increases SREBP DNA Binding—As a transcription
factor, SREBP-1 binds to specific sequences, i.e. SRE (5⬘-ATCACCCCAC-3⬘). To examine the effect of HMGB1 on the DNA
binding ability of SREBP-1, electrophoretic mobility shift assay(EMSA) was conducted using recombinant His-SREBP-1c and32
P-labeled DNA probe with SRE sequence. As shown in Fig.
6b, a single band with retarded migration by SREBP-1c wasdetected (lane 2). The specificity of this binding was confirmedby its supershift with anti-SREBP-1 antibody (lane 3) and bylack of binding of GST-HMGB1 alone to the probe (lane 4).
When GST-HMGB1 fusion protein was added, the intensities ofbands were enhanced in a dose dependent manner (lanes 5–7),
but not by GST alone (lane 8), indicating that HMGB1 stimulated the formation of SREBP-1䡠SRE complex. It was also remarkable that GST-HMGB1 did not change the mobility, suggesting that HMGB1 was not included in the SREBP-1䡠SRE
complex. The same results as above were obtained with
SREBP-2 (Fig. 7). These data demonstrate that HMGB1 promotes the binding of SREBP to SRE sequence.
To examine which HMGB1 domain contributes to this activation, we performed EMSA using deletion derivatives of
HMGB1 (Fig. 8a). Addition of recombinant full-length HisHMGB1 protein enhanced the formation of SREBP-1䡠SRE complex in a dose-dependent manner, whereas addition of HisHMGB1-Abox and -⌬Abox did not (Fig. 8b). This suggests that
A and B boxes are both required to promote the formation of the
SREBP䡠SRE complex.

FIG. 4. GST pull-down assays to determine the HMGB1 binding site of SREBP-1. a, schemes of functional domains of nuclear SREBP-1cand deletion derivatives were shown. b, schematic representation of a GST-HMGB1 fusion protein used in our pull-down assays. c– e, 35S-labeled
recombinant SREBP-1c proteins of various length generated by in vitro transcription/translation system were pulled down by GST-HMGB1 fusion
protein. Proteins pulled down as well as 4% of input were electrophoresed and visualized by BAS2000. Truncated proteins devoid of helix-loop-helix
domain were shown not to be pulled down. f, schematic representation of homology between SREBP-1 and -2. Helix-loop-helix domain shows thehighest similarity of 77% among their domains. g, Full-length nuclear forms of both SREBP-1 and -2 were demonstrated to be pulled down by
GST-HMGB1.

HMGB1 Activates SREBP
Luciferase Reporter Analyses Demonstrating That HMGB1
Activates SREBP in Vivo—To estimate the impact of HMGB1SREBP interaction on the transcriptional activity of SREBP in
the cells, series of transfection experiments were performed
using a plasmid containing luciferase reporter gene driven by

FIG. 5. GST pull-down assays to map the SREBP-1 binding siteof HMGB1. a, schematic representation of domain structure of HMGB1
and full-length or truncated versions of His-tagged HMGB1 proteins. b,schema of GST-SREBP-1 fusion protein used in pull-down assays. c,series of 35S-labeled recombinant HMGB1 deletion derivatives were
pulled down by GST alone or GST-SREBP-1 fusion protein. FL, fulllength HMGB1; Abox, truncated HMGB1 protein containing A-box
alone; ⌬Abox, deleted HMGB1 protein lacking A box.

27529

SREBP-responsive promoter (pSRE-Luc) and HMGB1 expression and/or RNA interference (RNAi) plasmids. As shown in
Fig. 8 (a and b), when HMGB1 expression was increased byco-transfection of its expression plasmid, the transcriptional
activity of SREBP-1c or -2 as assessed through pSRE-Luc wassignificantly enhanced. Conversely, when endogenous HMGB1
expression was knocked down by RNAi, the SREBP activity
was reduced in parallel. Moreover, when the effect of RNAi wascountered by HMGB1 expression plasmid that is resistantagainst the RNAi because it does not contain the RNAi target
sequence we selected in the 3⬘ non-coding region, the SREBP
activity was partially restored. These functional studies demonstrate that HMGB1 potentiates the transcriptional activities
of SREBP in cells, indicating that HMGB1-SREBP interaction
is physiologically relevant in vivo.
HMGB1 Promotes SREBP Dimerization—As describedabove, we demonstrated that HMGB1 interacts with SREBPs
through their HLH domains. Because HLH domains are known
to play a crucial role in the homodimerization of SREBP-1 (30 –32), we examined the effect of HMGB1 on the dimerization of
SREBP-1 using the TR-FRET assay system. FRET from donor toacceptor fluorescence molecule can be observed when the twocome in close proximity of each other. We first tested the bindingof His-tagged full-length HMGB1, HMGB1-Abox, or HMGB1⌬Abox to GST-SREBP-1 with this system; HMGB1 proteins werelabeled with anti-His antibody conjugated with donor fluorescence (europium), whereas GST-SREBP-1 and GST alone werelabeled with anti-GST antibody conjugated with acceptor fluorescence (allophycocyanin), and thereafter both were mixed and
TR-FRET from donor to acceptor was measured on a fluorometer.
Reiterating the conclusion from the GST pull-down assay, FRET
was observed in the case of full-length HMGB1 or HMGB1-Abox,
whereas no FRET occurred with HMGB1-⌬Abox (Fig. 9a).
We next examined whether dimerization of SREBP-1 could
be detected by FRET between His-SREBP-1 and GSTSREBP-1, which were labeled with donor (europium) and acceptor (allophycocyanin) fluorescence-conjugated antibody, respectively. As shown in Fig. 9b, addition of HMGB1 increased
FRET between His-SREBP-1 and GST-SREBP-1, suggestingthat HMGB1 promotes the dimerization of SREBP-1.

FIG. 6. EMSAs demonstrating HMGB1 enhancement of SREBP binding to SRE oligonucleotides. a, Coomassie Brilliant Blue (CBB)
staining of recombinant His-tagged SREBP-1 and -2 proteins used in our EMSA. These proteins were expressed in E. coli and purified withnickel-bound resin. b and c, EMSA with SRE probe detecting recombinant SREBP-1 (b) and -2 (c) (each 200 ng). The 32P-labeled oligonucleotides
were incubated with the indicated recombinant proteins and/or antibody and resolved on a 4.6% polyacrylamide gel. Lanes 2 and 5–7 show
increasing amounts of GST-HMGB1 protein enhances DNA binding of SREBP in a dose-dependent manner.

27530

HMGB1 Activates SREBP

FIG. 7. EMSAs showing that fulllength HMGB1 is required to promote the formation of SREBP䡠SRE
complex. a, Coomassie Brilliant Blue
(CBB) staining (left panel) and Western
blotting with anti-His-tag antibody (rightpanel)
ofrecombinant
His-tagged
HMGB1 proteins used in this experiment.
b, EMSA with SRE probe and recombinant GST-SREBP-1 protein mixed withvarious length of HMGB1. The differential effects of full-length HMGB1 (leftpanel) versus HMGB1 containing A box
alone (middle panel) and HMGB1 lacking
A box (right panel) on DNA binding of
GST-SREBP-1 are shown.

FIG. 8. Luciferase reporter analyses demonstrating that HMGB1 activates SREBP in vivo. a and b, the effects of increase or decreasein HMGB1 amount on SREBP-1c (a) or -2 (b) transcriptional activities as assessed by luciferase reporter analysis. Human embryonic kidney 293
cells were transfected with pSRE-Luc (0.25 ␮g) and pcDNA3.1-SREBP-1c or -2 expression plasmid (0.025 ␮g) together with the indicated HMGB1
expression/RNAi plasmids (1.0 ␮g each). As an expression plasmid for HMGB1, pcDNA3.1-HMGB1 was used, whereas the control contains thesame amount of empty pcDNA3.1 plasmid. To decrease the endogenous HMGB1, short hairpin RNA expression plasmid (pENTR/U6-HMGB1),
designed to evoke RNA interference against HMGB1 mRNA at a locus in the 3⬘ non-coding region (therefore not interfering with mRNA expressedby pcDNA3.1-HMGB1), was co-transfected (control is the same amount of pENTR/U6-lacZ, a short hairpin RNA expression plasmid targeting for
lacZ). After a 24-h incubation, the luciferase activity was measured on a luminometer. *, significance at p ⬍ 0.01 by two-tailed Student’s t test
against control. Bars, ⫾ S.E.
DISCUSSION

Our present studies clearly demonstrate that SREBPs interact with HMGB1, which enhances their binding to cognate
DNA sequences and thereby transcriptional activity. HMGB1

is a very abundant chromatin-binding protein, acting as anarchitectural facilitator in a variety of DNA-related processes,
including transcription, replication, V(D)J recombination, andrepair (reviewed in Refs. 21 and 22). In fact, HMGB1 has been

HMGB1 Activates SREBP

27531

FIG. 9. TR-FRET assays demonstrating that HMGB1 promotes SREBP-1 dimerization. a, TR-FRET observed between europium andallophycocyanin, which were used to label His-HMGB1 and GST-SREBP-1, respectively, with their conjugated antibody. Full-length HMGB1 (FL,left panel) and HMGB1 with A box alone (Abox, middle panel) proteins were shown to provoke FRET signals, whereas HMGB1 lacking A box(⌬Abox, right panel) was not. b, TR-FRET between His-SREBP-1 and GST-SREBP-1 labeled with europium and allophycocyanin, respectively, wasmeasured in the presence or absence of HMGB1 (without any tags). Combination of His-SREBP-1 and GST-SREBP-1 in the presence of HMGB1(lane 8) exhibited significantly higher FRET signals. *, significance at p ⬍ 0.001 by Fisher’s test against any control. Time-resolved fluorescenceexited at 340 nm and detected at 665 nm with delay time of 50 ␮s was measured by cumulative counts of 1000 repeats. The results are expressed
as 1000 ⫻ (665 nm/615 nm). Bars, ⫾ S.E.

implicated in the regulation, via both activation and repression, of transcription. The mechanisms are considered to be byinteracting both with the basal transcription machinery (33)
and with individual transcription factors such as Hox proteins(34), p53 (35), NF-␬B (36), and steroid hormone receptors (37).
In the latter case, HMGB1 enhances their binding to the cognate DNA sites. Our current findings presented SREBP asanother example of HMGB1 interaction with transcription
factors.
In our series of experiments, we found out that the HLH
domain of SREBP-1 interacts with HMGB1. Because this domain is well known to be committed to the dimerization of
SREBP-1 (30 –32), we examined the effects of HMGB1 on theprocess, and obtained the intriguing result that HMGB1 promotes the dimerization of SREBP-1. It is tempting to speculatethat this effect might contribute to the enhanced binding of
SREBP to SRE, whereas it is generally considered that DNA
bending induced by HMGB1 facilitates the DNA-protein interaction (22, 38). These mechanisms are not exclusive and mightperhaps be cooperative.
Recently a new paradigm has been proposed that nuclear
architectural proteins like HMGB1 are involved in the fastswitching of gene expression (38). Supporting this, mutant
yeast lacking HMGB1-related proteins, the non-histone proteins NHP-6A and -6B are severely retarded in the activation ofseveral inducible genes (39, 40). In this context, it is very
intriguing that SREBPs, especially SREBP-1, play an impor-tant role in the induction of genes encoding lipogenic enzymes,
which are the most vigorously up-regulated in liver and adipose
tissue when animals are refed after starvation (26, 29). Ourpresent findings thus raise the possibility that HMGB1 might
be potentially involved in this dynamic regulation of lipogenic
genes.
It has been documented that homozygous knock-out mice ofthe Hmgb1 gene are born alive, but die within 24 h due tohypoglycemia (41). When glucose is given parenterally,
Hmgb1-deficient mice survive for several days. These mice arereported to be completely lacking in adipose tissue. This phenotype of lipodystrophy might be related to its interaction with
SREBPs, because SREBPs have been proposed to be also involved in another type of lipodystrophy caused by lamin A/C
deficiency, where the interaction of SREBP-1 and lamin A/C isreported (42). Although further studies are needed, our present
finding that SREBPs interact with HMGB1 might provide goodclues for exploring the molecular mechanisms regulating lipogenesis and adiposity.
Our data demonstrated that SREBP-1 binds to HMGB1
through its HLH domain, which leads us to speculate thatmany other HLH-type transcription factors could interact with
HMGB1. In fact, we have already found that TFE3, the member closest to SREBP in the B-HLH-ZIP family, binds to
HMGB1 by Far Western analysis (data not shown), and othermembers might also interact with HMGB1. Investigation ofthese interactions between HMGB1 and HLH proteins will give27532

HMGB1 Activates SREBP

valuable insights into yet undiscovered roles of HMGB1 in theregulation of transcription.
Acknowledgments—We appreciate Alyssa H. Hasty for critical reading of the manuscript, Yoshiya Oda and Hiroyuki Arai for technicaladvice for mass spectrometry, and Kengo Miyagai and Taro Nakazawa
for assistance of TR-FRET assay.
REFERENCES
1. Brown, M. S., and Goldstein, J. L. (1997) Cell 89, 331–340
2. Brown, M. S., and Goldstein, J. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96,
11041–11048
3. Brown, M. S., Ye, J., Rawson, R. B., and Goldstein, J. L. (2000) Cell 100,391–398
4. Hua, X., Yokoyama, C., Wu, J., Briggs, M., Brown, M., Goldstein, J., and Wang,
X. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11603–11607
5. Bennett, M. K., Lopez, J. M., Sanchez, H. B., and Osborne, T. F. (1995) J. Biol.
Chem. 270, 25578 –25583
6. Ericsson, J., Jackson, S. M., Lee, B. C., and Edwards, P. A. (1996) Proc. Natl.
Acad. Sci. U. S. A. 93, 945–950
7. Shimano, H., Horton, J. D., Shimomura, I., Hammer, R. E., Brown, M. S., andGoldstein, J. L. (1997) J. Clin. Invest. 99, 846 – 854
8. Horton, J. D., Shimomura, I., Brown, M. S., Hammer, R. E., Goldstein, J. L.,
and Shimano, H. (1998) J. Clin. Invest. 101, 2331–2339
9. Sato, R., Okamoto, A., Inoue, J., Miyamoto, W., Sakai, Y., Emoto, N., Shimano,
H., and Maeda, M. (2000) J. Biol. Chem. 275, 12497–12502
10. Amemiya-Kudo, M., Shimano, H., Hasty, A. H., Yahagi, N., Yoshikawa, T.,
Matsuzaka, T., Okazaki, H., Tamura, Y., Iizuka, Y., Ohashi, K., Osuga, J.,
Harada, K., Gotoda, T., Sato, R., Kimura, S., Ishibashi, S., and Yamada, N.
(2002) J. Lipid Res. 43, 1220 –1235
11. Magana, M. M., Koo, S. H., Towle, H. C., and Osborne, T. F. (2000) J. Biol.
Chem. 275, 4726 – 4733
12. Oliner, J. D., Andresen, J. M., Hansen, S. K., Zhou, S., and Tjian, R. (1996)
Genes Dev. 10, 2903–2911
13. Sanchez, H. B., Yieh, L., and Osborne, T. F. (1995) J. Biol. Chem. 270,
1161–1169
14. Ericsson, J., Jackson, S. M., and Edwards, P. A. (1996) J. Biol. Chem. 271,
24359 –24364
15. Bennett, M. K., Ngo, T. T., Athanikar, J. N., Rosenfeld, J. M., and Osborne,
T. F. (1999) J. Biol. Chem. 274, 13025–13032
16. Ericsson, J., Usheva, A., and Edwards, P. A. (1999) J. Biol. Chem. 274,
14508 –14513
17. Zeng, L., Lu, M., Mori, K., Luo, S., Lee, A. S., Zhu, Y., and Shyy, J. Y. (2004)
EMBO J. 23, 950 –958
18. Rae, F. K., Martinez, G., Gillinder, K. R., Smith, A., Shooter, G., Forrest, A. R.,
Grimmond, S. M., and Little, M. H. (2004) Oncogene 23, 3067–3079
19. Misawa, K., Horiba, T., Arimura, N., Hirano, Y., Inoue, J., Emoto, N., Shi-mano, H., Shimizu, M., and Sato, R. (2003) J. Biol. Chem. 278,
36176 –36182
20. Yamamoto, T., Shimano, H., Nakagawa, Y., Ide, T., Yahagi, N., Matsuzaka, T.,
Nakakuki, M., Takahashi, A., Suzuki, H., Sone, H., Toyoshima, H., Sato, R.,
and Yamada, N. (2004) J. Biol. Chem. 279, 12027–12035
21. Bustin, M. (2002) Sci. STKE 2002, E39
22. Thomas, J. O., and Travers, A. A. (2001) Trends Biochem. Sci. 26, 167–174
23. Sheng, Z., Otani, H., Brown, M. S., and Goldstein, J. L. (1995) Proc. Natl. Acad.
Sci. U. S. A. 92, 935–938
24. Shimano, H., Horton, J. D., Hammer, R. E., Shimomura, I., Brown, M. S., andGoldstein, J. L. (1996) J. Clin. Invest. 98, 1575–1584
25. Shimano, H., Shimomura, I., Hammer, R. E., Herz, J., Goldstein, J. L., Brown,
M. S., and Horton, J. D. (1997) J. Clin. Invest. 100, 2115–2124
26. Shimano, H., Yahagi, N., Amemiya-Kudo, M., Hasty, A. H., Osuga, J., Tamura,Y., Shionoiri, F., Iizuka, Y., Ohashi, K., Harada, K., Gotoda, T., Ishibashi,S., and Yamada, N. (1999) J. Biol. Chem. 274, 35832–35839
27. Amemiya-Kudo, M., Shimano, H., Yoshikawa, T., Yahagi, N., Hasty, A. H.,
Okazaki, H., Tamura, Y., Shionoiri, F., Iizuka, Y., Ohashi, K., Osuga, J.,
Harada, K., Gotoda, T., Sato, R., Kimura, S., Ishibashi, S., and Yamada, N.
(2000) J. Biol. Chem. 275, 31078 –31085
28. Briggs, M., Yokoyama, C., Wang, X., Brown, M., and Goldstein, J. (1993)
J. Biol. Chem. 268, 14490 –14496
29. Horton, J. D., Bashmakov, Y., Shimomura, I., and Shimano, H. (1998) Proc.
Natl. Acad. Sci. U. S. A. 95, 5987–5992
30. Ferre-D’Amare, A. R., Prendergast, G. C., Ziff, E. B., and Burley, S. K. (1993)
Nature 363, 38 – 45
31. Parraga, A., Bellsolell, L., Ferre-D’Amare, A. R., and Burley, S. K. (1998)
Structure 6, 661– 672
32. Rishi, V., Gal, J., Krylov, D., Fridriksson, J., Boysen, M. S., Mandrup, S., andVinson, C. (2004) J. Biol. Chem. 279, 11863–11874
33. Ge, H., and Roeder, R. G. (1994) J. Biol. Chem. 269, 17136 –17140
34. Zappavigna, V., Falciola, L., Helmer-Citterich, M., Mavilio, F., and Bianchi,
M. E. (1996) EMBO J. 15, 4981– 4991
35. Jayaraman, L., Moorthy, N. C., Murthy, K. G., Manley, J. L., Bustin, M., andPrives, C. (1998) Genes Dev. 12, 462– 472
36. Brickman, J. M., Adam, M., and Ptashne, M. (1999) Proc. Natl. Acad. Sci.
U. S. A. 96, 10679 –10683
37. Boonyaratanakornkit, V., Melvin, V., Prendergast, P., Altmann, M., Ronfani,L., Bianchi, M. E., Taraseviciene, L., Nordeen, S. K., Allegretto, E. A., and
Edwards, D. P. (1998) Mol. Cell. Biol. 18, 4471– 4487
38. Bianchi, M. E., and Beltrame, M. (2000) EMBO Rep. 1, 109 –114
39. Moreira, J. M., and Holmberg, S. (2000) EMBO J. 19, 6804 – 6813
40. Fragiadakis, G. S., Tzamarias, D., and Alexandraki, D. (2004) EMBO J. 23,
333–342
41. Calogero, S., Grassi, F., Aguzzi, A., Voigtlander, T., Ferrier, P., Ferrari, S., andBianchi, M. E. (1999) Nat. Genet. 22, 276 –280
42. Lloyd, D. J., Trembath, R. C., and Shackleton, S. (2002) Hum. Mol. Genet. 11,
769 –777