Synergy among Nuclear Receptor Coactivators: Selective Requirement for Protein Methyltransferase and

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Molecular and Cellular Biology, June 2002, p.
3621-3632, Vol. 22, No. 11
Synergy among Nuclear Receptor Coactivators
Selective Requirement for Protein
Methyltransferase and Acetyltransferase
Activities
Young-Ho Lee,1 Stephen S. Koh,2 Xing Zhang,3
Xiaodong Cheng,3 and Michael R. Stallcup1,2
Departments of Pathology,2 Biochemistry and
Molecular Biology, University of Southern
California, Los Angeles, California 90089,1
Department of
Biochemistry, Emory University School of
Medicine, Atlanta, Georgia 303223
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2
Hypothetical model for transcriptional
activation by ER, mediated by three coactivators,
GRIP1, p300, and CARM1
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Nuclear receptors (NR)
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comprise a family of transcription factors that
regulate gene expression in a ligand-dependent
manner. Members of the NR superfamily include
receptors for steroid hormones, such as estrogens
(ER) and glucocorticoids (GR), receptors for
nonsteroidal ligands, such as thyroid hormones
(TR) and retinoic acid (RAR), as well as
receptors that bind diverse products of lipid
metabolism, such as fatty acids and
prostaglandins. contain a highly conserved DNA
binding domain (DBD) in the central region of the
polypeptide chain that binds specific enhancer
elements in the promoters of target genes.
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p160 coactivators (NR coactivators)
Proteins of 160 kDa molecular mass were among
the first factors identified that interact with
NRs in a highly ligand-dependent, both in
solution or on DNA. These biochemically
identified factors could themselves associate
with CBP/p300. bind directly to activated NR and
recruit a variety of secondary coactivators. SRC-1
, GRIP1 and ACTR..
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Acetyltransferase
CBP/p300, p/CAF can acetylate histone, nonhistone
protein and have been associated with chromatin
remodeling. contribute to transcriptional
activation through multiple molecular
mechanism. associate with coactivator with
protein acetyltransferase activity. interact
with components of the basal transcription
machinery and thus may help to recruit the
transcription preinitiation complex to the
promoter
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Methyltransferase
CARM 1 (PRMT4) coactivator-associated
arginine methyltransferase 1(Histone H3) PRMT
1 protein arginine methyltransferase 1 (Histone
H4) Most histone methylation occurs on lysine,
though arginine methylation also occurs on
histones H3 and H4 Lysine methylation is highly
selective, with the best-characterized sites
being K4 and K9 of histone H3. In general, K9
methylation is associated with transcriptionally
inactive heterochromatin, while K4 methylation is
associated with transcriptionally active
euchromatin (Boggs et al., 2002 Litt et al.,
2001 Nakayama et al., 2001 Nishioka et al.,
2002a ). Protein arginine methylation has been
implicated in signal transduction, nuclear
transport and transcription regulation. Protein
arginine methyltransferases (PRMTs) mediate the
AdoMet-dependent methylation of many proteins,
including many RNA binding proteins involved in
various aspects of RNA processing and/or
transport.
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Sites and Structures of Methylated Residues
in Histone Tails (A) Known sites of methylation
in the N-terminal tails of histones H3 and H4.
Lysines found to be methylated are shown in red
and arginines are shown in green. Lysines have
the potential to be mono-, di-, or trimethylated.
Known histone methyltransferases and their
preferred methylation sites are also
indicated. (B) Chemical structures of lysine and
its methylated derivatives. The action of histone
methyltransferases (HMTs) is indicated. The
potential reversibilty of the reaction by
demethylases is indicated with question
marks. (C) Chemical structures of arginine and
its methylated derivatives. The action of histone
methyltransferases (HMTs) is indicated. The
potential demethylation reactions are indicated
with question marks. The two forms in which
dimethylarginine can be found are symmetric or
asymetric.
Cell, Vol 109, 801-806, 28 June 2002
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Structural features of arginine
methyltransferases and information on their
activity.
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(100 ng)
(10 ng)
FIG. 1. Requirement for three coactivators
(GRIP1, CARM1, and p300) at low
levels of NR.
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FIG. 2. Ternary coactivator complex formation
among GRIP1, CARM1 and p300/CBP. (A)
Coimmunoprecipitation. (B) Modified mammalian
two-hybrid system. (C) Coactivator assays.
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FIG. 3. Synergy among various combinations of
three or four coactivators at low
NR levels.
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FIG. 4. Role of protein acetyltransferase
activities of p/CAF and p300 in coactivator
synergy. WT, wild-type p/CAF or p300 MT1, p/CAF
?579-608 MT2, p/CAF ?609-624 MT, p300
?1603-1653 ,present.
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FIG. 5. Role of protein methyltransferase
activity of CARM1 in coactivator synergy. (A)
Methyltransferase activities of wild-type and
mutant CARM1. (B) Coactivator synergy with
different amounts of wild-type and mutant CARM1.
(C) Coactivator activities of mutant
and wild-type CARM1 at low versus high NR levels.
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FIG. 6. Selective synergy of protein arginine
methyltransferases with p300 and p/CAF.
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• At low NR levels,
• NR alone failed to activate the reporter gene,
  even when hormone was present
• Expression of all three coactivators resulted in
  a strong synergistic enhancement of NR function
  and in fact was required for efficient NR
  function
• The methyltransferase activity of CARM1 was
  required for the coactivator synergy and thus for
  reporter gene activation
• At high NR levels,
• NR alone activated the reporter gene in a
  hormone-dependent manner
• The activity achieved by adding all three
  coactivators was no more effective, and was often
  less effective, than the activity observed with
  two coactivators
• The methyltransferase activity of CARM1 was not
  required for its ability to cooperate with GRIP1
  to enhance NR activity

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Different mechanisms of transcriptional activation
Low NR levels
High NR levels
Different mechanisms of transcriptional
activation at low and high NR levels. (Left) At
low NR levels, NRs shuttle on and off of the
hormone response element (HRE), and occupancy of
the HRE is relatively infrequent. Transcriptional
activation by the bound NRs requires the
assistance of multiple coactivators to remodel
chromatin structure and recruit and activate RNA
polymerase II (Pol II complex). (Right) High NR
levels may force almost constant occupancy of the
HREs by NRs. Perhaps such high occupancy allows
NRs to recruit RNA polymerase through direct
contact with TATA binding protein (TBP), TFIIB,
or other components of the RNA polymerase II
complex, independent of the action of some or
many coactivators.