Department of Biochemistry and Molecular Biology, Program in Genetics, Michigan State University, East Lansing, MI 48824-1319, USA
* Author for correspondence (e-mail: arnosti{at}msu.edu)
Accepted 30 September 2003
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SUMMARY |
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Key words: Enhancer, Cis-regulatory element, Enhanceosome
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Introduction |
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Such integrative functions have been ascribed to the human
interferon-ß (IFN-ß) enhancer, which drives transcription of the
IFN-ß gene in response to viral infection
(Struhl, 2001). The presence
of each transcription factor binding site and its precise arrangement within
the regulatory element are critical for the various regulatory proteins
(sequence-specific activators and architectural proteins) to assemble through
cooperative interactions into a well-defined nucleoprotein complex called the
`enhanceosome'. Assembly of the enhanceosome is essential for the
transcription of the IFN-ß gene in response to viral infection in cells.
In this structured element, the exact arrangement of factor binding sites is
critical to dictating the output of the element, so the enhanceosome acts as a
molecular computer, leading to a single output directed to the general
machinery (Thanos and Maniatis,
1995
; Kim and Maniatis,
1997
; Munshi et al.,
2001
). Such a complex might provide a stereospecific interface for
interaction with the basal transcriptional machinery, possibly engaging
several components of the basal machinery simultaneously to effect synergistic
activation (Carey et al., 1990
;
Chi et al., 1995
). With such
an enhancer, the target gene would be activated only upon the assembly of a
`complete' complex, providing a precise on/off binary transcriptional switch
in response to the appropriate stimulus.
Studies of developmentally regulated genes have also provided examples of
enhancers as molecular computers. The developmentally regulated Drosophila
even-skipped (eve) gene is regulated by developmental enhancers
that are thought to act in a computational fashion. The reiterated stripe
pattern of eve expression in the blastoderm embryo is generated by
modular enhancers bound by broadly expressed transcriptional activators and
regionally distributed repressors (Fujioka
et al., 1999; Small et al.,
1992
; Small et al.,
1996
). These enhancers interpret gradients of regulatory factors
and are active or inactive, depending on the particular set of regulatory
proteins present in a given nucleus. The eve stripe 2 enhancer is
active only in a narrow band of cells where activators Bicoid and Hunchback
are present, but repressors Krüppel, Giant and Sloppy-paired are scarce
or absent (Andrioli et al.,
2002
; Small et al.,
1992
). Key to the functional autonomy of the modular eve
enhancers is the short-range of the repressors that regulate individual
enhancers; for example, the short-range transcriptional repressor Krüppel
bound to the stripe 2 enhancer in central regions of the embryo does not
interfere with the activity of the adjacent eve stripe 3 enhancer
(Small et al., 1993
). An
assumption is that each enhancer works as a single computational unit, not a
redundant set of independently acting elements. Consistent with this view is
the finding that enhancer function is disrupted upon loss of a single
activator or repressor site (Arnosti et
al., 1996a
; Small et al.,
1992
). However, these experiments have relied on minimal elements
that may already represent a subset of the actual regulatory region (see
Discussion).
A more detailed picture emerges from the functional dissection of the
endo 16 cis-regulatory region of Strongylocentrotus
purpuratus. The endo 16 gene is regulated during development by
a 2.3 kb region containing binding sites for factors that contribute to
distinct functions such as early widespread activation, late activation,
repression of the early element, and potentiation of the repressor sites.
Separate portions of the regulatory region can be combined to recreate some or
all of the expression pattern, and models based on Boolean logical operators
successfully simulate the output of these regulatory regions
(Yuh et al., 1998). These
studies emphasize the integrative, computer-like processing suggested to be a
characteristic of developmental enhancers, and suggest that basal elements
respond to signals generated by these molecular logic circuits.
In contrast to this view of the enhancer as an information-processing unit, we find that a single, compact enhancer can serve as an information display, representing on and off states, at the same time and in the same nucleus. This finding suggests that rather than acting as a computer that integrates various inputs, enhancers can simultaneously display both the active and repressed states, which may be interpreted by successive or multiple, simultaneous interactions with the basal transcriptional machinery. In this case, the enhancer does not act in a concerted, computational fashion, and the basal transcriptional machinery plays an active, rather than a passive, role in interpreting signals from the enhancer.
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Materials and methods |
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Reporter genes
The plasmid UAS-lacZ (Brand and
Perrimon, 1993) was modified to contain two Giant sites
(5' GGC CGC TAT GAC GCA AGA AGA CCC AGA TCT TTT TAT GAC GCA
AGA GA 3') or two Knirps sites (5'GGC CGC ATC TGA
TCT AGT TTG TAC TAG ACA TCT GAT CTA GTT TCA 3') 20
nucleotides upstream of the five Gal4 binding sites. The resulting vectors
named M2g5u-lacZ or M2k5ulacZ
(Fig. 1C,D) respectively,
consist of two Giant or Knirps binding sites, five tandemly arrayed Gal4
binding sites, followed by the Hsp70 TATA box and transcriptional
start driving lacZ expression. These reporters were further modified
by introducing oligos containing two Twist and two Dorsal binding sites
(Szymanski and Levine, 1995
)
at the NotI site upstream of the Giant or Knirps sites resulting in
the 2twi.dl-M2g5u-lacZ and 2twi.dl-M2k5u-lacZ reporters
(Fig. 1A,B,E,F).
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Results |
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Simultaneous repression and activation
When Gal4 activators are combined with Dorsal and Twist activators on a
composite element, strongly enhanced staining is noted in the central regions
of the embryo, indicative of additive or synergistic activation. In the
regions of the embryo containing the repressors Giant or Knirps, the width of
the area in which the nuclei are stained (a 18-20 swathe of nuclei) is the
same as the pattern of staining driven by the Gal4 protein alone. We conclude
that in nuclei containing Giant and Knirps protein, the pattern of staining
directed by Dorsal and Twist is being selectively repressed by the short-range
repressors, while transcription driven by Gal4 (a narrower 18-20 nuclei
swathe) is unimpeded (Fig.
1E,F). The pattern of gene expression indicates that, in nuclei
where the activators and repressors are co-expressed, transcription is driven
by one cluster of activators within the compact regulatory element, while at
the same time other activators within the same element are being actively
repressed by Giant or Knirps. This compact regulatory element therefore, has
subelements that represent both `active' and `inactive' states simultaneously,
unlike the binary switch activity observed for many enhancers, where it
appears that a single signal to activate or repress is present.
We make this conclusion based on the activity of the elements when only one
set of activators is present (Fig.
1A-D), and on the characteristic narrower pattern driven by the
Gal4 activators. Consistent with this conclusion, a similar pattern of
exclusive repression of the Dorsal and Twist activators is seen when
expression of Gal4 is driven in a ubiquitous pattern using the nanos
promoter (Tracey et al.,
2000). Here, we can compare promoter activity with Gal4 alone or
in combination with Dorsal/Twist (Fig.
1G). In dorsal regions of the embryo, the only activator on the
element is Gal4, and no repression by Giant is visible. In the ventral regions
where Dorsal and Twist are present, but the repressor is absent, more intense
staining is seen, consistent with synergistic or additive activation.
Importantly, in the ventral regions also containing the Giant repressor
(Fig. 1G, arrows),
lacZ expression is similar to that observed in the dorsal regions of
the embryo. This result indicates that Dorsal and Twist are not working
together with Gal4, but are functionally independent and selectively repressed
in the regulatory element.
Compact element functions in a distance-, orientation-,
promoter-independent manner
To further evaluate the properties of this element, we tested whether it
possessed classical characteristics of a transcriptional enhancer, namely,
acting in a distance- and orientation-independent manner
(Banerji et al., 1981). The
element containing Giant binding sites was placed in either orientation
between the divergently transcribed white (at -265 bp) and
lacZ (at -130 bp) genes. In both orientations tested, this element
directed white expression from -265 bp in a manner closely resembling
that seen for the Hsp70 lacZ reporter; Giant efficiently repressed
Dorsal and Twist, while Gal4 activated transcription in a continuous ventral
swathe (Fig. 1B,F,
Fig. 2A,B,D,E). A similar
pattern of repression and activation is seen with the transposase
lacZ gene (Fig. 2C,F). The
identical results observed in Fig.
1F and Fig.
2B,C,E,F indicate that the specific patterns of activation and
repression are not dependent on the particular promoter context or orientation
of activators and repressors.
Conversion of enhancer output to a binary on/off state
The compact regulatory element assayed in Figs
1 and
2 fits the classic definition
of an enhancer, functioning in a distance- and orientation-independent manner.
In addition, the size of this element resembles that of naturally occurring
enhancers 200-800 bp in length. However, the element does not function in
the biphasic either `on or off' mode, normally thought to be a characteristic
feature of enhancers. We are unaware of documented cases where a single
enhancer displays two different states at the same time and in the same
nucleus, thus this dual activity appears to be unusual. It is possible that
rather than being an inherent functional property of enhancers, the uniform
output of enhancers might reflect evolutionary pressure to arrange repressor
and activator binding sites to optimize a consistent output. To simulate this
situation, two additional Giant repressor binding sites were introduced into
this element 3' of the Gal4 binding sites. Now, complete loss of
staining is evident in nuclei containing the Giant protein (arrows) yielding a
classic biphasic `on or off' state (Fig.
3A-D).
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Discussion |
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Functional analyses of cis-regulatory regions provide evidence for
redundancy and hence divisibility, of natural enhancers, suggesting that they
can also contain multiple, independently acting subelements. In the viral
setting, the well-studied SV40 enhancer comprises two independently acting
subelements that can be separately assessed
(Herr and Clarke, 1986). In
Drosophila, recent evidence suggests that eve enhancers
possess redundant activities. Deletion of the entire 480 bp eve
stripe 2 element within the eve locus fails to completely abrogate
stripe 2 expression (M. Kreitman, personal communication) indicating the
presence of redundant regulatory sequences in the locus. Furthermore,
tissue-specific expression of the yolk protein genes yp1 and
yp2, is supported by flanking sequences after deletion of the 125 bp
yolk protein enhancer (Piano et al.,
1999
). The resilience of natural enhancers to loss of single
binding sites further supports the notion that these elements are built of
redundantly acting sequences (Arnosti,
2003
).
Selection for uniformity of enhancer output
A scenario of an enhancer with simultaneously displayed activation and
repression states is reminiscent of the modular, autonomous pair-rule stripe
enhancers, such as even-skipped stripe elements, where separate
enhancers represent different `states' of repression and activation in the
same nucleus (Gray and Levine,
1996). An important distinction is that our findings suggest that
a similar discrimination is taking place within the tight confines of a single
enhancer, and that in order to establish a uniform signal output, enhancers
require a proper stoichiometry or distribution of repressor and activator
binding sites to ensure that all possible enhancer subelements provide the
same information (Fig. 4). Indeed a distributed pattern of short-range transcriptional repressor binding
sites is typical of many developmental enhancers that function in the early
Drosophila embryo; this configuration would allow repressors to block
multiple modes of enhancer-promoter interactions
(La Rosee et al., 1997
;
Small et al., 1992
;
Small et al., 1996
). In this
study we actually measure the simultaneous independent activity of
sub-elements (Figs 1,
2) and show that they can be
deployed to give a unitary response (Fig.
3) as is seen with natural enhancers. Thus, the carefully designed
internal organization of cis-regulatory modules can provide uniform
information that closely simulates an integrative information processing
capacity.
|
With such flexibility, the transcription factors of an enhancer might still engage the transcriptional machinery in simultaneous cooperative interactions, as is suggested with enhanceosomes. However, our studies suggest that an individual enhancer is capable of representing both the state of activation and repression, suggesting that the basal machinery may `sample' discrete regions, consisting of a small number of transcription factor binding sites, within the enhancer (Fig. 4B,C). Successive interactions with the basal machinery, and the biochemical consequence of these multiple interactions would dictate the overall output of the enhancer (Fig. 4B). Alternatively the enhancer may engage in multiple, simultaneous contacts with some or all of the enhancer bound proteins, with repressors such as Giant and Knirps preventing some of these interactions (Fig. 4C). In either case, multiple iterative sampling of the enhancer, or simultaneous readout, the enhancer would function as an information display element with computation at the level of enhancer-promoter interactions.
Our results suggest that a closer examination of enhancer classifications
is warranted. The terms enhancer and enhanceosome are frequently used
interchangeably to denote a complex of DNA-bound regulatory proteins, yet
there appear to be important functional distinctions between enhanceosomes, as
typified by the IFN-ß enhancer, and other regulatory elements. In the
light of the functional differences outlined above, a distinction should be
made between the terms enhanceosome, which requires the cooperative
assembly of a higher order structure within an enhancer, and other
cis-regulatory elements that may or may not function in this manner. We
propose a model, the information display or `billboard' model for enhancer
action, in which an enhancer, rather than acting as a central processing unit,
can display contrasting information, which is then interpreted by basal
transcriptional machinery (Fig.
4B,C). The binary `on or off' decisions that appear to be
transmitted by the enhancer to the basal machinery actually result from the
basal machinery reading a series of redundant signals encoded within the
enhancer. The model does not explicitly describe the molecular mechanisms of
repression and activation, but direct contacts between the Drosophila
activators used here and components of the basal machinery are supported by
biochemical studies (Koh et al.,
1998; Pham et al.,
1999
; Yuh et al.,
1998
; Zhou et al.,
1998
)
The billboard enhancer model appears to more accurately describe many
developmentally regulated enhancers, whose internal architecture is subject to
rapid evolutionary change, even as the overall output remains constant
(Ludwig et al., 2000;
Ludwig et al., 1998
). Although
studies such as those on the IFN-ß gene indicate that cells may commonly
use enhanceosomes to achieve regulatory precision in gene expression, it is
likely that eukaryotic organisms use the `billboard' type of enhancers to
achieve diversity in gene expression patterns and evolutionary
flexibility.
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ACKNOWLEDGMENTS |
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