Structure, Function, and Activator-Induced Conformations of the CRSP Coactivator

Abstract

The human cofactor complexes ARC (activator-recruited cofactor) and CRSP (cofactor required for Sp1 activation) mediate activator-dependent transcription in vitro. Although these complexes share several common subunits, their structural and functional relationships remain unknown. Here, we report that affinity-purified ARC consists of two distinct multisubunit complexes: a larger complex, denoted ARC-L, and a smaller coactivator, CRSP. Reconstituted in vitro transcription with biochemically separated ARC-L and CRSP reveals differential cofactor functions. The ARC-L complex is transcriptionally inactive, whereas the CRSP complex is highly active. Structural determination by electron microscopy (EM) and three-dimensional reconstruction indicate substantial differences in size and shape between ARC-L and CRSP. Moreover, EM analysis of independently derived CRSP complexes reveals distinct conformations induced by different activators. These results suggest that CRSP may potentiate transcription via specific activator-induced conformational changes.

Full text

Structure, Function, and
Activator-Induced
Conformations of the CRSP
Coactivator
Dylan J. Taatjes,
1
Anders M. Na¨a¨r,
1
Frank Andel III,
1,2
Eva Nogales,
1,2
Robert Tjian
1
*
The human cofactor complexes ARC (activator-recruited cofactor) and CRSP
(cofactor required for Sp1 activation) mediate activator-dependent transcrip-
tion in vitro. Although these complexes share several common subunits, their
structural and functional relationships remain unknown. Here, we report that
affinity-purified ARC consists of two distinct multisubunit complexes: a larger
complex, denoted ARC-L, and a smaller coactivator, CRSP. Reconstituted in vitro
transcription with biochemically separated ARC-L and CRSP reveals differential
cofactor functions. The ARC-L complex is transcriptionally inactive, whereas the
CRSP complex is highly active. Structural determination by electron microscopy
(EM) and three-dimensional reconstruction indicate substantial differences in
size and shape between ARC-L and CRSP. Moreover, EM analysis of indepen-
dently derived CRSP complexes reveals distinct conformations induced by
different activators. These results suggest that CRSP may potentiate transcrip-
tion via specific activator-induced conformational changes.
Initiation of eukaryotic transcription is reg-
ulated at multiple stages by mechanisms
involving activators, core promoter recog-
nition complexes, and chromatin modifying
factors (1, 2). Once assembled at their cog-
nate DNA sites, sequence-specific enhanc-
er binding proteins typically rely upon var-
ious types of coactivators to communicate
activation signals to the preinitiation com-
plex, which consists of transcription factors
IIA (TFIIA), TFIIB, TFIID, TFIIE, TFIIF,
TFIIH, and RNA polymerase II. One large
class of eukaryotic transcriptional coactiva-
tors is characterized by the ability to poten-
tiate transcription via interactions with
activators and/or the basal transcription
apparatus. Among this diverse group of
transcriptional cofactors are multisubunit
complexes such as yeast Mediator, as well
as a cadre of related metazoan coactivators,
which include the ARC/DRIP, TRAP/
SMCC, hMed, NAT, CRSP, and PC2 com-
plexes (3–11). These metazoan complexes
contain a few subunit homologs of yeast
Mediator proteins, whereas the majority
of their subunits appear to have diverged
considerably, likely reflecting the greater
complexity of metazoan gene regulatory
pathways.
The various mammalian cofactor com-
plexes can be grouped into two general
categories consisting of a set of larger
(ARC/DRIP, TRAP/SMCC, hMed, and
NAT) and smaller (CRSP and PC2) cofac-
tors. Whereas these “large” and “small”
cofactors share many subunits, the larger
complexes contain additional polypeptides
(ARC240, ARC250, cdk8, and cyclin C)
not present in the smaller CRSP and PC2
complexes. This distinguishing structural
feature has not been clearly linked to dis-
tinct functional characteristics. In fact, both
cofactor subclasses (large and small) gen-
erally display coactivator function in in
vitro transcription assays, although NAT
and SMCC may mediate a form of repres-
sion (3–5, 7–11). Here, we present evi-
dence that correlates distinguishing struc-
tural features with transcriptional function
in the context of the ARC (large) and CRSP
(small) cofactors. In addition, our structural
analysis indicates that an aspect of coacti-
vator (CRSP) function may involve specific
activator-induced conformational changes.
We initiated EM structural studies on an
ARC cofactor fraction purified from HeLa
nuclear extract as described previously (8). A
phosphocellulose 1.0 M KCl eluate was used
to isolate ARC via a VP16 affinity resin (8);
the eluted ARC sample was then applied to a
glycerol gradient. Subsequent classification
of ARC particle images in electron micro-
graphs of negatively stained samples showed
that the affinity-purified ARC preparation
contained two complexes of distinct size and
shape, present in approximately a 60:40 ratio
(small:large; Fig. 1A). Similar results were
obtained using ARC complexes isolated via
an SREBP-1a (8) affinity resin (12). To better
separate the two complexes, the affinity-pu-
rified ARC sample was subjected to “higher-
resolution” glycerol gradient sedimentation
in which the sample represented ⬍5% of the
total gradient volume. This typically sedi-
mented the small complex in fraction 13,
whereas the large complex was concentrated
in fraction 17. SDS–polyacrylamide gel elec-
trophoresis (SDS-PAGE) (Fig. 1B) and west-
ern blot analysis (12) confirmed that the
small complex was highly related (or identi-
cal) to the previously identified CRSP coac-
tivator (10), whereas the large complex pos-
sessed a subunit composition characteristic of
the ARC/DRIP and TRAP/SMCC complexes
(4, 5, 8, 9). Thus, previous ARC preparations
(8) most likely contained two distinct, struc-
turally stable complexes. We will provision-
ally refer to the large complex as ARC-L
(ARC-Large) and the small complex as
CRSP. The “CRSP” designation is based pri-
marily on the presence of CRSP70, which
appears to be CRSP-specific (see below).
We next determined whether the differ-
ences in complex structure and subunit com-
position might reflect different transcriptional
cofactor properties. For this functional anal-
ysis, we used an in vitro–reconstituted tran-
scription system (13) comprising an LDLR-
derived template assembled into chromatin
by the S-190 system (14). This chromatin
template was then used to direct Sp1/SREBP-
dependent activation of transcription in a re-
action containing purified and recombinant
human transcription factors (TFIIA, TFIIB,
TFIID, TFIIE, TFIIH, TFIIF, and RNA poly-
merase II). When we tested the activity of
glycerol gradient fractions containing CRSP,
but little or no ARC-L, we observed robust
Sp1/SREBP-dependent activation (Fig. 2A,
lanes 7 and 8). Analysis of samples contain-
ing a mixture of CRSP and ARC-L revealed
that as the ratio of ARC-L/CRSP increased,
there was a concomitant decrease in tran-
scriptional activity (Fig. 2A, lanes 10, 12, and
14). Note, however, that some activation still
occurred at the higher ARC-L ratio (lane 14).
This was because glycerol gradient purifica-
tion, although effective in yielding CRSP
fractions largely free of ARC-L, was not able
to generate ARC-L fractions completely free
of CRSP (gradient fractions most concentrat-
ed in ARC-L still contained ⬃30 to 40%
CRSP, based on statistical analysis of EM
data). Therefore, to obtain an ARC-L sample
devoid of CRSP, we immunodepleted these
fractions with an antibody to CRSP70 (anti-
CRSP70), which recognizes only the CRSP
complex [see supplementary information
(15)]. When CRSP was immunodepleted
from these ARC-L fractions, there was a
1
Howard Hughes Medical Institute and
2
Lawrence
Berkeley National Laboratory, Department of Molec-
ular and Cell Biology, 401 Barker Hall, University of
California, Berkeley, CA 94720, USA.
*To whom correspondence should be addressed. E-
mail: jmlim@uclink4.berkeley.edu
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dramatic loss of coactivator activity (Fig. 2A,
lane 16), indicating that highly purified
ARC-L is unable to potentiate transcriptional
activity in this assay. By contrast, when glyc-
erol gradient fractions were exhaustively de-
pleted of ARC-L by anti-cdk8 immunodeple-
tion, there was no detectable decrease in tran-
scriptional activity (Fig. 2A, compare lanes 6
and 8), consistent with our previously report-
ed findings (8). As an additional control, we
isolated CRSP by an entirely independent
method using an anti-Flag resin to immuno-
purify the CRSP complex from a cell line
expressing Flag-tagged CRSP70 (16). This
“Flag-CRSP” complex (eluted with Flag pep-
tide) also strongly potentiated activator-de-
pendent transcription (Fig. 2A, lane 4). The
CRSP complex isolated via Flag-CRSP70
immunopurification exhibited a subunit com-
position that was indistinguishable from glyc-
erol gradient affinity-purified CRSP as deter-
mined by immunoblot and silver stain analy-
sis (12).
To further confirm these findings, we
immunopurified CRSP and ARC-L from
the P1M fraction using antibodies directed
against CRSP70 and cdk8. Each separate
immunoprecipitated complex was tested for
transcriptional activity by supplementing
the reconstituted transcription reaction with
cofactor-bound affinity beads. Activator-
dependent transcription was observed for
the CRSP70 immunoprecipitated complex
(Fig. 2B, lanes 3 and 4); in contrast, resins
containing only the ARC-L complex (␣-
cdk8) were essentially inactive (Fig. 2B,
lanes 1 and 2). A “beads only” mock im-
munoprecipitate was used as a negative
control (Fig. 2B, lanes 5 and 6) and showed
no activity. These functional assays indi-
cate that the ARC250, ARC240, cdk8, and
cyclin C subunits present in ARC-L may
somehow render the complex inactive, at least
in this LDLR-derived promoter context.
Structural analyses were performed with
the same ARC-L and CRSP samples used
for the functional assays. Samples were
analyzed in negative stain (uranyl acetate)
using EM and single-particle image recon-
struction techniques. Further details regard-
ing image processing are provided as sup-
plementary information (15). The three-di-
mensional (3D) structure of ARC-L is
shown in Fig. 3A. Defined “body,” “leg,”
and “foot” regions are clearly distinguish-
able and are consistent with the structural
features of the TRAP complex, which has
similar subunit composition (17). The leg
contacts the foot at one point and the body
at two sites, giving rise to a hole halfway
along the length of the cofactor. The foot
region is oriented at a right angle to the
body, giving the complex an “L” shape as
shown by the side (orientation 3) view of
the complex in Fig. 3A.
Structural studies of CRSP were initiat-
ed using a VP16 affinity-purified sample.
The VP16-CRSP structure possesses three
distinct regions, including a hook-like
“leg” domain and a central “body” connect-
ed by two contacts to a “head” region (Fig.
3B). The complex is quite elongated and
narrow (360 Å by 145 Å) and contains
holes at each end. Comparison of the VP16-
CRSP structure with ARC-L (also isolated
via VP16) reveals important structural re-
lationships. A superposition of the com-
plexes, on the basis of their related struc-
Fig. 1. Activators bind two distinct cofactor
complexes. (A) Structural separation of the
complexes by image processing. VP16 affini-
ty-purified “ARC” was analyzed by EM. A
total of 2027 particles and their correspond-
ing tilt pairs were windowed, aligned, and
merged into 26 classes. Two-dimensional av-
erages representing two of these classes are
shown, indicative of the different-sized com-
plexes observed in the affinity-purified ARC
sample (bar, 150 Å). (B) Subunit composi-
tions of small (CRSP) and large (ARC-L) com-
plexes. SDS-PAGE (5 to 15%) silver stain
analysis indicates presence of CRSP70 specif-
ically in CRSP and ARC240, ARC250, cdk8,
and cyclin C specifically in ARC-L. Presence
(or lack) of many subunits have been corrob-
orated by western blot (12). Asterisks denote
nonspecific bands that do not consistently
copurify with the CRSP complex. Data shown
utilized an SREBP-1a affinity-purification
step. Analogous results were obtained using
VP16 affinity-purification.
Fig. 2. CRSP and ARC-L have contrasting transcriptional cofactor properties. (A) In vitro
transcription analysis of ARC-L and CRSP on an LDLR-derived chromatin template. Transcrip-
tion reactions were performed in the presence (⫹) and absence (–) of SREBP-1a (5 nM) and Sp1
(2 nM). Reactions were supplemented with CRSP and ARC-L as shown. Lanes 1 and 2: no
cofactor added. Lanes 3 and 4: CRSP isolated via Flag-tagged CRSP70 (peptide eluted). Lanes
5 and 6: affinity-purified (SREBP) CRSP immunodepleted of ARC-L via ␣-cdk8. Lanes 7 and 8:
affinity-purified (SREBP) CRSP. Lanes 9 through 14: affinity-purified ( VP16) samples containing
mixtures of CRSP and ARC-L. Lanes 15 and 16: affinity-purified ( VP16) ARC-L immunodepleted
of CRSP via ␣-CRSP70. Equal amounts of cofactor(s) were added to each transcription reaction
on the basis of silver stain analysis (12). (B) In vitro transcription reactions of immunopre-
cipitated complexes. Reactions were executed as in (A), except that added cofactor complexes
were bound to protein A beads via the antibody indicated. Equal amounts of the immunopre-
cipitated complexes were added to each transcription reaction on the basis of silver stain
analysis (12).
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tural features, is depicted in Fig. 3C. Both
possess similar head/body and leg regions;
in fact, the shape and location of the hole in
the leg region of VP16-CRSP and (VP16)
ARC-L is virtually identical. However, the
CRSP complex apparently lacks some pro-
tein density in its head/body region and is
completely devoid of the foot domain
present in ARC-L. This is highlighted by
the corresponding difference map (ARC-L
minus CRSP) shown in Fig. 3D. Figure 3E
shows the location of the VP16 activator
binding site on the CRSP complex, on the
basis of EM analysis and difference map-
ping of VP16-CRSP samples following in-
cubation with anti-GST [VP16 affinity pu-
rification utilizes a glutathione S-trans-
ferase (GST) fusion protein]. EM samples
of VP16-CRSP were prepared as described
above, followed by addition of the antibody
(in a fivefold excess). Two independent
experiments were run: one with a poly-
clonal and one with a monoclonal antibody
to GST. Both experiments yielded the same
result. Note that the location of the VP16
binding site is exposed in both the CRSP
and ARC-L complexes.
Because Flag-CRSP was essentially identi-
cal in function and subunit composition (Fig. 2)
(12) relative to VP16-CRSP, we presumed that
these independently purified complexes would
possess similar structural characteristics. How-
ever, EM analysis revealed that the Flag-CRSP
complex adopted a different conformation
(compare Fig. 4A and 4B). Although it retained
distinct head, body, and leg domains, the over-
all shape of the “activator-free” Flag-CRSP
structure was wider (180 Å versus 145 Å),
shorter (300 Å versus 360 Å), and flatter (130 Å
versus 150 Å) than the VP16-CRSP structure.
Further, it appeared that the second bridge be-
tween the head and body region was displaced
to a more central location in the Flag-CRSP
conformer (Fig. 4, A and B; compare structures
in row 1).
Having obtained two quite distinct struc-
tures for activator-free (Flag) CRSP versus ac-
tivator (VP16)– bound CRSP, we next exam-
ined the structure of CRSP bound to a different
activator, SREBP-1a. EM analysis of SREBP-
CRSP revealed a third distinct conformation
(Fig. 4C) with few structural similarities to
Flag-CRSP and even fewer to VP16-CRSP
(compare structures in Fig. 4). Although the
length and width (305 Å by 180 Å) of SREBP-
CRSP was similar to Flag-CRSP, it possessed
greater structural variation in its head and body
regions, appearing considerably more hollow in
its center. This resulted in the complex being a
bit “broader” (165 Å versus 130 Å) (Fig. 4, B
and C; compare structures in row 2) with re-
spect to Flag-CRSP. In addition to the qualita-
tive comparisons in Fig. 4, cross-correlation
[detailed in supplementary information (15)] of
the structures demonstrates that VP16-CRSP,
Flag-CRSP, and SREBP-CRSP are conforma-
tionally distinct. Figure 4D shows the region
(highlighted in yellow) likely to contain the
SREBP binding site. This site was identified
Fig. 3. Structural analysis of ARC-L and CRSP. Three-dimensional reconstruc-
tion of (A) ARC-L and (B) CRSP at 32 Å resolution. Complexes are rendered to
2.0 MD (ARC-L) and 1.25 MD (CRSP; based on 0.81 dalton/Å
3
conversion), the
approximate predicted molecular weights of the cofactors. Dimensions are
shown. Rotation of the volumes 180° gives the second orientation (opposite
face of the complex—row 2). Additional rotation by 90° provides a third
“side” view of the cofactors. Both complexes were isolated with a VP16
affinity-purification step. (C) Overlay of CRSP structure (orange outline) on
ARC-L showing the proposed structural relationships between the complexes.
(D) Difference map (ARC-L minus CRSP) showing regions of extra protein
density in ARC-L. Protein density in the difference map corresponds to 700
kD, the approximate molecular weight difference between CRSP and ARC-L.
(E) Localization of VP16 activation domain binding site (yellow) on the CRSP
coactivator (see text).
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from EM analysis and difference mapping of
structures generated by incubation of SREBP-
CRSP with anti-GST antibodies, which target
the GST–SREBP-1a activator fusion (8). As
with the VP16 localization experiments (see
above), both polyclonal and monoclonal anti-
bodies were used in two independent analyses,
both of which generated the same result.
To provide additional evidence for activa-
tor-induced structural alterations in CRSP, we
selectively converted activator-free Flag-CRSP
to either the VP16 or SREBP conformation.
This was done by first affinity-purifying Flag-
CRSP on an anti-Flag affinity column. Then,
either SREBP-1a or VP16 (8) was allowed to
bind the immobilized Flag-CRSP prior to pep-
tide elution from the anti-Flag affinity res-
in. Subsequent EM analysis and 3D recon-
struction revealed that the predicted activa-
tor-induced conformational changes had
occurred. The Flag-CRSP sample which
bound VP16 structurally resembled the
VP16 affinity-purified complex shown in
Fig. 4A; similarly, Flag-CRSP which
bound SREBP was converted to a confor-
mation that corresponded specifically with
Fig. 4C. Cross-correlation analysis further
established the integrity of the conforma-
tional changes. The 3D structures and cor-
relation analysis of these Flag-converted
structures are included with the supplemen-
tary information (15). As with previous EM
analyses, each reconstruction was conduct-
ed completely independently for both sam-
ples without reference bias. On the basis of
the conformational consistency and repro-
ducibility shown by these independent
structural comparisons (Fig. 4) (15), we
conclude that the CRSP coactivator is con-
formationally flexible and can assume sig-
nificantly different 3D structures when
bound to different activators.
The ARC and CRSP cofactor complexes,
originally purified from human cells by inde-
pendent means, presented a dilemma with re-
gard to their structural and functional relation-
ships. In this study, we used a combination of
biochemical separation methods, in vitro tran-
scription assays, and EM-based structural anal-
ysis to resolve potential functional and structur-
al differences. These studies reveal three unex-
pected findings. First, previously defined ARC
preparations (8) actually consist of two distinct
and stable multisubunit complexes, identified
as ARC-L and CRSP. Second, functional anal-
yses of the ARC-L and CRSP complexes reveal
that the larger ARC-L complex is transcription-
ally inactive, whereas the smaller CRSP com-
plex displays potent coactivator function in
vitro. Third, and perhaps most surprisingly, EM
analysis and 3D reconstruction of CRSP, either
not bound to ligand or bound to two different
activation domains, reveals three distinct struc-
tures. This third finding suggests that CRSP
may undergo substantial conformational al-
terations induced by binding different activa-
tors. Such activator-induced structural chang-
es may have a profound impact on the mech-
anism of transcriptional activation in vivo
and highlights the potential importance of
coactivator structural plasticity in the forma-
tion of transcriptionally active preinitiation
complexes.
Although CRSP and ARC-L both bind ac-
tivators with high affinity, they display con-
trasting transcriptional properties. This suggests
that one mechanism for transcription regulation
may involve modulation of transcriptional ac-
tivity by the ARC-L–specific subunits. The
presence of ARC240, ARC250, and cdk8/cy-
clin C may halt transcription by favoring for-
mation of the inactive ARC-L complex (with
concomitant loss of CRSP70), as shown in Fig.
5. Such a model is supported by our in vitro
transcription assays; the NAT and SMCC co-
factors, which have subunit compositions sim-
ilar to ARC-L, have also displayed transcrip-
tional inactivity in other in vitro assays (5, 11).
Given that a prominent difference between
ARC-L and CRSP is the presence of ARC240,
ARC250, and cdk8/cyclin C, it is likely that
these subunits play a negative role in transcrip-
tional regulation. Indeed, cdk8 is known to
inhibit RNA polymerase II elongation by
blocking TFIIH-mediated phosphorylation of
the RNA polymerase CTD (18). Conversely,
the CRSP70 subunit may play an important role
in the coactivator function of the CRSP com-
plex. This is suggested by its conspicuous ab-
sence in the inactive ARC-L cofactor. Addi-
tionally, CRSP70 contains a region highly ho-
mologous to TFIIS, suggesting it may mediate
a key interaction with RNA polymerase II (19).
EM analysis and 3D reconstruction of
ARC-L and CRSP revealed important struc-
tural relationships. The extra protein density
in ARC-L appears to reside in both the head
and the foot domains, suggesting that the
additional ARC-L components do not bind as
a single “subcomplex.” The predicted mass
of the foot domain is 330 kD. The combined
mass of cdk8/cyclin C is about 90 kD; thus,
the foot region of ARC-L most likely con-
tains (at least) one of the ARC240 or
ARC250 polypeptides.
Structural analysis of the CRSP coactivator
reveals that activator binding induces dramatic
structural changes in the complex, which ap-
pear to be activator-specific. One reason for the
observed activator-specific changes may be that
the VP16 and SREBP-1a activation domains
target different subunits in the CRSP complex
[CRSP77 versus ARC105(TIG-1), respective-
ly] (20, 21) and these distinct activator-targeted
subunits may act as a “switch” to orchestrate
specific conformational changes. The differenc-
es in conformation cannot be attributed to the
Fig. 4. Structures of (A)
VP16-CRSP, (B) Flag-
CRSP, and (C) SREBP-
CRSP show substantial
differences in confor-
mation. Each complex is
rendered to 1.25 MD,
which approximates
their predicted molecu-
lar masses. Dimensions
are as shown; structures
have been filtered to 32
Å resolution. Structures
across each row (1 and
2) show the same rela-
tive orientation. (Note:
The relative orienta-
tions of the complexes
cannot be known with
absolute certainty be-
cause each structure
was generated indepen-
dently via random coni-
cal tilt.) Complexes in
row 2 are rotated 90°
with respect to row 1,
showing the side view
of the coactivator. (D)
Localization of SREBP
binding site (yellow) on the CRSP coactivator (see text).
The white arrow indicates the approximate location of
the VP16 binding site on the opposite face (back) of the
complex, based on the results in Fig. 3E. The SREBP-
CRSP orientation shown is the face opposite that shown
in (C), row 1.
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mere presence of the activation domain because
of its small size (about 6% of the total mass)
relative to the CRSP complex. Further, we have
mapped the VP16 and SREBP-1a binding sites
to comparatively small and distinct regions on
the CRSP complex. This provides direct evi-
dence that only a limited number (one or per-
haps two) of CRSP subunits are targeted by a
particular activator. Because VP16-CRSP and
SREBP-CRSP are conformationally distinct in
regions distal to the activator binding sites, we
suggest that activator binding may induce long-
range conformational changes. Thus, different
protein surfaces in CRSP are likely exposed as
a consequence of activator binding.
The conformational flexibility of the
CRSP coactivator may have important impli-
cations for its mechanism of action. CRSP
and its related coactivator complexes appear
to be generally required for transcription and
are targeted by a diverse array of regulatory
proteins (22). Interestingly, different tran-
scription activators can target different sub-
units of the CRSP complex (3, 9, 20, 21).
Thus, despite binding the same coactivator
complex, regulatory proteins may impart pro-
moter-specific functions that may be depen-
dent on CRSP conformation. For example,
specific activator-induced CRSP conforma-
tions may regulate binding and recruitment of
additional activators or cofactors to the
preinitiation complex (Fig. 5). Furthermore,
these conformational changes may trigger
other (as yet undiscovered) enzymatic activ-
ities within the CRSP coactivator. Indeed,
adopting a number of activator-dependent
conformations may enable CRSP to perform
more specialized roles in transcriptional acti-
vation. Elucidation of these roles will be an
important subject of future work.
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Shelton, and K. Yamamoto for critical reading of
the manuscript. We also thank A. Ladurner and S.
Ryu for the flag-70 cell line; A. Ladurner, S. Ryu,
and W. Zhai for antibodies against CRSP70; Y.
Nedialkov and S. Triezenberg for monoclonal anti-
bodies against GST; and O. Fedin for scanning
micrographs. D.J.T. is supported by a grant from
the American Cancer Society (#PF0007801GMC).
This work was funded by grants from the NIH and
Howard Hughes Medical Institute.
9 August 2001; accepted 7 December 2001
Linking Breeding and Wintering
Ranges of a Migratory Songbird
Using Stable Isotopes
D. R. Rubenstein,
1,2
*†C. P. Chamberlain,
2
‡R. T. Holmes,
1
M. P. Ayres,
1
J. R. Waldbauer,
2
‡G. R. Graves,
3
N. C. Tuross
4
We used the natural abundance of stable isotopes (carbon and hydrogen) in the
feathers of a neotropical migrant songbird to determine where birds from
particular breeding areas spend the winter and the extent to which breeding
populations mix in winter quarters. We show that most birds wintering on
western Caribbean islands come from the northern portion of the species’ North
American breeding range, whereas those on more easterly islands are primarily
from southern breeding areas. Although segregated by breeding latitude, birds
within local wintering areas derive from a wide range of breeding longitudes,
indicating considerable population mixing with respect to breeding longitude.
These results are useful for assessing the effects of wintering habitat loss on
breeding population abundances and for predicting whether the demographic
consequences will be concentrated or diffuse.
In recent decades, many species of neotropi-
cal migrant birds have shown marked chang-
es in abundance—both increases and de-
creases—in parts of their North American
breeding range (1,2). These changes may be
due to events occurring in the breeding
?
CRSP
GTFs
CRSP
Activator
CRSP
70
?
ARC-L
subunits
ARC-L
SREBP
GTFs
GTFs
"VP16"
Fig. 5. Model showing proposed mechanism by
which CRSP and ARC-L regulate transcription.
In this model, activators play a dual role in
transcriptional activation. First, they recruit
the CRSP coactivator to the promoter. Second,
they induce conformational changes in the
CRSP complex, which may facilitate transcrip-
tion initiation by recruiting/stabilizing other
cofactors and components of the preinitiation
complex, including RNA polymerase II. Certain CRSP interactions may be activator-specific.
However, upon binding additional ARC-L subunits (which may occur on activator-bound CRSP
following multiple rounds of activated transcription), CRSP undergoes a structural change that may
also result in dissociation of CRSP70. Now converted to ARC-L, coactivator function is lost and
activated transcription is inhibited. The location of ARC-L–specific polypeptides (red) is based upon
analysis detailed in Fig. 3D. The orientations of the complexes at the promoter are speculative.
VP16 is shown in quotation marks because it does not directly bind DNA.
REPORTS
8 FEBRUARY 2002 VOL 295 SCIENCE www.sciencemag.org1062