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First published online December 7, 2008
doi: 10.1242/10.1242/dev.030064
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, 701 West 168th Street, New York, NY 10032, USA.
* Author for correspondence (e-mail: or38{at}columbia.edu)
Accepted 22 October 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: C. elegans, Neuronal development, Left/right asymmetry, Transcriptional regulation
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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). Initially characterized by morphological criteria,
more modern functional, physiological and molecular approaches have revealed
an astounding diversity of cell types in the nervous system, more so than in
any other organ (Masland,
2004; Nelson et al.,
2006). In order to understand the development of an organ composed
of such vastly diverse cell types, it is useful to study the development of
the simple units that make up the organ. One approach to breaking down the
nervous system into simple units is the hierarchical clustering of neurons
into groups of neurons that share common features such as morphology or
function (`neuron classes' or `neuron types') and further clustering these
groups into subtypes or subclasses. These subtypes share fundamental common
functions or morphologies, but they differ in a restricted subset of features
that define individual subtypes.
How are the regulatory mechanisms responsible for neuron type and neuron
subtype specification integrated? Specifically, are the regulatory factors
that control neuron type specification completely separate from those that
control ensuing subtype specification or are they mechanistically linked? We
address this here by exploring the specification of the bilaterally symmetric
ASE neuron class in the nematode C. elegans. This neuron class is
composed of two neurons, each defining its own subtype: the left ASE neuron
(ASEL) and the right ASE neuron (ASER). Both neuron subtypes are
morphologically and synaptically indistinguishable and share a large battery
of co-expressed genes, but display differential asymmetric expression of a
class of putative chemoreceptor genes (reviewed by
Hobert et al., 2002).
ASE neuron class specification is controlled by the terminal selector gene
che-1 (Chang et al.,
2003; Etchberger et al.,
2007; Uchida et al.,
2003). che-1 encodes a zinc-finger transcription factor
that directly controls the expression of its target genes by binding to its
cognate binding sequence: the ASE motif. It is the expression of these target
genes, including ion channels, neurotransmitter receptors and
neuropeptide-encoding genes, that characterizes the terminally differentiated
bilateral ASE neurons. While adopting their bilaterally symmetric terminal
fate, the ASE neurons execute a further subtype diversification program in
which the left and right ASE neurons diversify by differentially expressing
distinct members of a family of putative chemoreceptors, encoded by the
gcy genes (Fig. 1A)
(Ortiz et al., 2006;
Yu et al., 1997). This
diversification program involves a gene regulatory loop, composed of
transcription factors and miRNAs (Fig.
1A) (Johnston et al.,
2005), and results in the asymmetric expression of the
gcy genes. The expression of components of this regulatory loop, as
well as the gcy genes themselves, is lost in che-1 mutants
and each of these genes contains ASE motifs in their promoters that are
required for their expression in ASE
(Etchberger et al., 2007).
Therefore, CHE-1 not only determines terminal differentiation features of ASE,
but also directly induces downstream regulatory events required for subtype
specification.
The necessity of CHE-1 for the specification of both neuronal subtypes,
ASEL and ASER, poses the question of specificity. How does CHE-1 induce solely
ASEL-specific genes in ASEL and ASER-specific genes in ASER, even though CHE-1
is expressed in both ASEL and ASER, and is able to induce bilaterally
expressed genes in both neurons? Genetic evidence has shown that differential
expression of genes in the regulatory loop shown in
Fig. 1A is required for ASE to
adopt either ASEL or ASER fate, but exactly how this mechanism functions at
the level of cis-regulatory motifs is unknown. We envision several distinct
models that are addressed here in this paper and are schematically presented
in Fig. 1B. Model 1 is based on
the observation that initially after the birth of ASEL and ASER, the two ASE
subtypes appear to share the expression of terminal differentiation markers
that eventually become restricted to ASEL or ASER during late embryogenesis
and early larval development (Johnston et
al., 2005). Model 1 posits that CHE-1 is required only for the
induction of the bilateral precursor state, inducing the expression of both
terminal markers (such as gcy-7, which is initially expressed in both
ASEL and ASER) and regulatory factors that directly control ASEL or
ASER-specific genes. These regulatory factors may then be sufficient to induce
the L/R diversification, thereby making CHE-1 activity superfluous after this
initial specification event (Fig.
1B). In Model 2, CHE-1 is required throughout the lives of the ASE
neurons and actively participates in controlling the L/R asymmetry of the gene
expression profiles in these cells. As CHE-1 acts in a target gene-specific
manner in both ASEL and ASER, the L/R specificity cannot be brought about
simply by directly modulating intrinsic CHE-1 activity (e.g. by
post-transcriptional mechanisms such as protein modifications or stability).
Rather, L/R specificity would need to be brought about by the repression of
CHE-1 activity in a target gene-dependent manner through what we term here
`co-repressor' molecules (Fig.
1B, Model 2a). Alternatively, CHE-1 may not be sufficient to
activate L/R-restricted target genes and may require what we term here
`co-activator' molecules (Fig.
1B, Model 2b).
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). As indicated
in the figure legends, constructs were injected either as circular subcloned
DNA or as a linear PCR fragment, generated either by PCR fusion or PCR
amplification of a cloned product. DNA constructs were injected into an
otIs151 background, which expresses a ceh-36::DsRed2
transgene to facilitate the identification of expression of gfp-based
reporter genes in ASEL/R. The gut-specific elt-2::gfp reporter (a
gift from J. McGhee) was used as an injection marker. Mutagenesis reactions
were performed using the QuickChange II XL Site Directed Mutagenesis Kit
(Stratagene). A list of DNA sequences can be provided on request.
RNAi assays
RNAi by feeding was performed (see
Ahringer, 2005) in the
nre-1 lin15b sensitized background
(Schmitz et al., 2007). For
scoring of F1s, 10-15 staged L3/L4 hermaphrodites were placed onto plates
seeded with dsRNA-expressing clones and incubated for 5 days at 22°C
before scoring the F1 progeny as adults on a Zeiss axioplan microscope. In the
case of P0 scoring, 100-150 staged L1s were placed on the plates and incubated
for 3-4 days before scoring as adults.
Electrophoretic mobility shift assay (EMSA)
CHE-1 and CEH-36 proteins were expressed and purified, and binding
reactions were performed (see Etchberger et
al., 2007). For quantitative cold competition assays, different
types of cold competitor (ASE motifs from genes tested) were added at specific
ratios relative to the labeled ceh-36 probe to the
protein/ceh-36 probe mixture after 10 minutes of the initial binding
reaction and allowed to incubate with the protein/probe complex for an
additional 10 minutes before loading on the gel. After detection of bound
probe using the Molecular Dynamics PhosphorImager and quantification with
ImageJ, the fraction of bound labeled ceh-36 probe compared with
bound uncompeted probe was plotted against the molar ratio of unlabeled to
labeled probe (Glass et al.,
1988). The affinities of each probe relative to the
ceh-36 probe were calculated by comparing the slopes of a plot of
[(1-Y)/Y] versus amount of competitor, where Y is the fraction of bound
labeled probe at a given ratio of unlabeled to labeled probe
(Glass et al., 1988).
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) (data not shown). To assess whether the function of
che-1 function is restricted to the initiation of ASE differentiation
or extends to the maintenance of the differentiated state
(Fig. 1B, Model 1 versus Model
2), we reduced che-1 activity in a temporally restricted manner by
performing RNAi on transgenic animals expressing either an ASEL-specific
marker (lim-6::gfp) or an ASER-specific marker (gcy-5::gfp).
The ASE neurons undergo their final division at 
380 minutes
post-fertilization (Sulston et al.,
1983). che-1 expression can be observed as early as one
cell division before the birth of ASEL and ASER, and persists through
adulthood (B. Tursun and O.H., unpublished). We delivered che-1 dsRNA
by feeding animals at the larval L1 stage (i.e. long after ASE has been
generated in mid-embryogenesis). If che-1 activity was required
solely for the initiation of gene expression in ASEL/R, then only the F1
progeny of RNAi-treated animals and not the P0 animals should show a loss or
reduction of gfp expression. If che-1 activity was
continuously required during the lifetime of the animal, the removal of
che-1 would result in a loss or reduction of gfp expression
in the acutely RNAi-treated P0 animals. After treatment of staged L1 animals
with che-1 RNAi, we observed a significant reduction of both
ASEL-specific and ASER-specific marker expression in both ASEL and ASER in
adult P0 animals (Fig. 2A,B).
These results indicate that che-1 is required not only for the
initiation but also for the maintenance of gene expression in ASEL/R.
Testing cis-regulatory models for the control of L/R asymmetric gene expression
The continuous requirement of che-1 activity for ASEL/R gene
expression means that L/R asymmetric transcription of terminal differentiation
genes necessitates a mechanism that restricts the activity of bilaterally
expressed CHE-1 in a target gene-dependent (i.e. promoter-dependent) manner to
either ASEL or ASER. `Promoter-dependent' means that bilaterally expressed
genes respond to CHE-1 in both ASEL and ASER, whereas L/R asymmetrically
expressed genes respond only to CHE-1 in either ASEL or ASER. As mentioned in
the Introduction, this may involve either promoter-dependent co-activator or
co-repressor mechanisms (Fig.
1B). To test these models, we analyzed the cis-regulatory
architecture of four asymmetrically expressed genes that are direct targets of
che-1, as they each contain ASE motifs required for expression in the
ASE neurons (Etchberger et al.,
2007; Sarin et al.,
2007): two putative chemoreceptors of the guanylyl cyclase family,
ASEL-expressed gcy-7 and ASER-expressed gcy-5
(Yu et al., 1997); the
ASEL-expressed LIM-homeodomain transcription factor lim-6
(Hobert et al., 1999); and the
ASEL-expressed microRNA lsy-6
(Johnston and Hobert, 2003).
The activation model (Fig. 1B,
Model 2b) predicts that the ASE motifs of each of these genes are not alone
sufficient to promote expression in both ASE neurons, whereas the repression
model (Fig. 1B, Model 2a)
predicts that the ASE motif alone can drive expression in both ASE neurons. We
find that each isolated ASE motif from the four asymmetrically expressed genes
when fused to gfp produces reporter gene expression in both ASE
neurons (Fig. 3). As this
bilateral expression is lost in the context of larger regulatory elements,
these regulatory elements must therefore contain information that somehow
restricts CHE-1 and ASE motif activity. To dissect the nature of the
additional regulatory information, we undertook a systematic deletion analysis
of the L/R asymmetrically expressed regulatory regions of these genes
(Fig. 4).
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). We first created seven non-overlapping deletion
windows of 22-32 bp in length (Fig.
4A). Previous results have shown that the
che-1-responsive ASE motif of the gcy-7 promoter lies at the
junction of deletion windows 3 and 4, the only deletions that resulted in loss
of expression in ASEL (Etchberger et al.,
2007). Of the seven deletion windows, three constructs (del2, del5
and del7) show a partially penetrant derepression of gfp expression
in ASER (Fig. 4A; see Figs S1
and S2 in the supplementary material). A triple deletion (del2,5,7) does not
further increase the penetrance of de-repression, even though the levels of
de-repressed gfp appear to be somewhat increased
(Fig. 4A; see Fig. S2 in the
supplementary material). These findings support the `co-repressor model' in
Fig. 1B.
To refine our mapping of the repressive regulatory architecture, we
generated smaller deletions within the identified repressive sequences. For
all three regions, our analysis revealed discrete repressive sequences 6-8 bp
in length that recapitulated the derepression effects of the larger deletions
(del2b; del4b; del7a) (Fig. 4A;
see Fig. S2 in the supplementary material). We also deleted small
phylogenetically conserved motifs within the ASE motif-containing region del3
(del3a) (see Fig. S3 in the supplementary material) and deletion window 6
(del6a) (see Fig. S3 in the supplementary material). As a result of both
deletions, we observed partial derepression of gfp in ASER
(Fig. 4A; see Fig. S2 in the
supplementary material). None of the four small motifs show notable
similarities to one another, but each is phylogenetically conserved in the
gcy-7 promoter of three other nematode species (see Fig. S3 in the
supplementary material). One of the four motifs, del4b, matches a predicted
NK-2 homeodomain binding site (TAAGTT)
(Noyes et al., 2008). Other
ASEL-specific gcy genes have some, but not all, of these motifs. We
deleted two of these motifs (TTCGGGAG and GGAAAAAA) in gcy-14 and
gcy-20, but these deletions did not affect ASEL-specific expression
of these genes (data not shown).
We noted a predicted K50-homeodomain binding site (TAATCC) directly downstream of the ASE motif. Deletion of this motif (del4a) from the gcy-7 minimal promoter resulted in a loss of gfp expression in ASEL. This is surprising as this deletion construct retains its ASE motif, which, like any other ASE motif tested, drives bilateral expression in complete isolation (Fig. 3). In the context of the gcy-7 promoter, the CHE-1-binding ASE motif, therefore, needs to collaborate with a `co-activator' motif (Fig. 1B, Model 2b) to activate ASEL-specific expression.
The `co-activator' motif also confers a left/right asymmetric activity to the ASE motif as a construct that contains only the ASE motif and the neighboring co-activator motif is asymmetrically expressed, with gfp levels in ASEL being substantially higher than in ASER (Fig. 4A) (compare ASEgcy-7#1 with ASEgcy-7#2). In other words, such a minimal motif is derepressed in ASER owing to the loss of the repressor motifs described above, but it still retains L/R asymmetry to some degree.
Intriguingly, the loss of ASE expression observed upon loss of the co-activator motif (del4a) is completely suppressed if the del4a deletion is combined with deletions of the three repressor motifs: del2, del5 and del7. The resulting quadruple mutant construct is expressed in a bilaterally symmetric manner in both ASEL and ASER (Fig. 4A). We interpret these findings to mean that the ASE motif of gcy-7 can, in principle, drive bilateral expression in ASEL and ASER; this activity alone is repressed through repressive cis-regulatory motifs, but the presence of the TAATCC motif can overcome this repression specifically in ASEL (Fig. 4A). In summary, gcy-7 is regulated by a combination of the `co-activator' and `co-repressor' models as shown in Fig. 1B.
Case 2: gcy-5
We started the analysis of the cis-regulatory region of the gcy-5
gene again with a scanning deletion analysis on the fragment of the
gcy-5 regulatory control region (gcy-5prom2, 306 bp),
which is required and sufficient to drive ASER-specific expression of a
reporter gene (Etchberger et al.,
2007). We deleted, in a non-overlapping manner, 24-31 bp windows
(Fig. 4B). Three of the 11
deletion windows (del3, del7 and del11) each resulted in a low penetrant
de-repression of gfp expression in ASEL, while leaving expression in
ASER unaffected (Fig. 4B; see
Figs S1 and S2 in the supplementary material). Double (del7,11) and triple
deletions (del3,7,11) exhibited an increase in the penetrance of de-repression
of gfp in ASEL (see Fig. S2 in the supplementary material). However,
neither the double nor triple deletion constructs showed equivalent bilateral
expression in ASEL/R. Bilateral expression is observed, however, if all
sequences around the ASE motif are chopped away, leaving only a 24 bp minimal
element (Fig. 3)
(Etchberger et al., 2007). We
conclude that repressive elements within region 3, 7 and 11 are, in part,
responsible for repression of gcy-5 expression in ASEL. Notably, in
contrast to the gcy-7 regulatory region, the gcy-5
regulatory region does not appear to contain a `co-activator' motif as no
mutation other than the ASE motif deletion results in a loss of reporter gene
expression.
To define individual sequence motifs within these repressive regions, we
created smaller deletions within each repressive region in an attempt to
recapitulate the derepression resulting from the larger individual deletions
(Fig. 4B; see Fig. S2 in the
supplementary material). For del3 and del7, we were able to identify short
sequences (8 bp and 7 bp, respectively), the loss of which resulted in
de-repression of gfp expression in ASEL. Neither motif shows
unambiguous matches to known transcription factor-binding sites predicted by
MatInspector (Cartharius et al.,
2005). For del11, we were unable to identify any discrete motif,
even though our smaller deletions targeted nearly the entire original deletion
window (Fig. 4B; see Fig. S2 in
the supplementary material).
The repressor motifs share little sequence similarity with each other and
are not conserved in C. briggsae. The C. briggsae gcy-5
promoter, when injected into C. elegans, does not drive detectable
gfp expression in ASE neurons (data not shown). This finding is also
consistent with our previous observations that, in spite of strong synteny of
at least some of the individual loci, the regulation of several other
gcy genes and also their genomic organization has significantly
diverged in C. briggsae (Ortiz et
al., 2006). Last, we also did not find the motifs conserved in
other ASER-restricted gcy genes, such as gcy-1 and
gcy-22.
Case 3: lim-6
Previous promoter analysis has identified a 200 bp minimal cis-regulatory
sequence of the lim-6 locus that is required and sufficient to direct
expression of a gfp reporter gene in ASEL
(Etchberger et al., 2007). We
generated eight non-overlapping deletions of 24-28 bp within the minimal
lim-6 promoter. Four out of the eight deletions (del4, del6, del7 and
del8) resulted in the loss of gfp expression in ASEL
(Fig. 4C)
(Etchberger et al., 2007). In
contrast to our analysis of gcy-5 and gcy-7, none of the
deletions resulted in the derepression of gfp in ASER.
The lim-6 minimal promoter contains two ASE motifs, one in
deletion window 6 and one in deletion window 7. The targeted deletion of each
of these ASE motifs individually resulted in a loss of gfp expression
(Etchberger et al., 2007).
However, as mentioned above, two additional deletion windows, del4 and del8,
also resulted in loss of gfp expression
(Etchberger et al., 2007). The
del4 and the del8 sequence share no similarity with the ASE motif consensus
sequence or with one another. They are also poorly conserved in other nematode
species (see Fig. S3 in the supplementary material).
To identify the positive-acting cis-regulatory motif(s) in each region, we generated sub-deletions. No sub-deletion in del8 recapitulated the effect of the complete deletion, but within del4, two adjacent deletions, del4a and del4d, resulted in a loss of gfp expression in ASEL (Fig. 4C). In contrast to the temporally persistent loss of expression observed from the del4 construct, both del4a and del4d displayed normal ASEL-specific expression up to early larvae stages (L1/L2), but not in later staged larvae or adults (Fig. 4C).
Taken together, the ASE motifs of lim-6, even though supporting bilateral expression in isolation, are prevented from doing so in a full promoter context and require additional co-activator motifs to support expression in ASEL.
Case 4: lsy-6
The 931 bp intergenic region between the lsy-6 miRNA hairpin and
its upstream gene contains all the regulatory information required for
ASEL-specific expression and function of the ASEL-fate inducer lsy-6
(Johnston and Hobert, 2003;
Sarin et al., 2007).
Successive deletions identified a 95 bp element (prom3) that is sufficient to
recapitulate expression only in ASEL (Fig.
4D). As the lsy-6 locus is highly conserved across four
nematode species (Fig. 4D), we
used sequence conservation as a guide for further deletion analysis. We
deleted four conserved sequence patches contained within the minimal 95 bp
regulatory element. Deletion of a putative E-box, a predicted bHLH
protein-binding site (del5; CANNTG) resulted in a complete loss of
gfp expression. Therefore, as in the case of gcy-7 and
lim-6, within its normal genomic context, the ASE motif (which
remains intact upon deletion of the E-box) is not sufficient to drive ASE
expression but requires an additional regulatory motif to be active. Moreover,
we found that deletion of another conserved motif, del3, resulted in a
completely penetrant de-repression of gfp expression in ASER
(Fig. 4D, see Fig. S2 in the
supplementary material) (gfp de-repression is only partially
expressive and cannot be further enhanced in a del3; del4 double deletion). No
obvious transcription factor-binding sites match this sequence, as assessed by
MatInspector (Cartharius et al.,
2005).
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Taken together, the restriction of ASE motif activity in the lsy-6 promoter appears most similar to that of gcy-7: each promoter contains repressive elements and in each case additional co-activator motifs are required for ASE motif expression. However, the identified cis-regulatory elements share no similarity to one another.
In conclusion, the four cases analyzed here demonstrate that the regulation of asymmetric gene expression in ASEL/R requires a remarkably distinct spectrum of cis-regulatory mechanisms that differ in a gene-specific manner. In each case, promoters contain an ASE motif that in complete isolation drives bilaterally symmetric expression in ASEL and ASER. In the context of its normal promoter, ASE motifs do not display this sufficiency. The activity of the ASE motif either becomes restricted by repressive cis-regulatory elements; or it becomes dependent on the presence of additional activating cis-regulatory motifs; or it becomes restricted by a combination of both co-activators and co-repressors. None of the isolated additional cis-regulatory elements displays any similarity to one another.
The CEH-36 Otx homeodomain protein binds to a L/R asymmetry-controlling motif
How are the co-activator and co-repressor motifs identified above
regulated? Previous genetic analysis has revealed a number of transcription
factors involved in determining L/R asymmetry
(Fig. 1A). However, little to
no information about possible binding sites of these transcription factors
exists, with the exception of the K50-homeodomain protein CEH-36, a C.
elegans OTX homolog and the only K50-homeodomain protein known to be
expressed in ASE (Lanjuin et al.,
2003). K50-homeodomain proteins bind to a TAATCC core consensus
sequence (Treisman et al.,
1989) and such a sequence is required for the L/R asymmetric
activation of gcy-7 in ASEL (Fig.
4A) (del4a) (see Fig. S3 in the supplementary material). Notably,
the mutant phenotype of ceh-36 is consistent with ceh-36
activating gcy-7 expression, as gcy-7 expression is lost in
ceh-36 mutants (Chang et al.,
2003). We find that bacterially produced CEH-36, but not two other
tested homeodomain proteins, can indeed bind to the TAATCC motif in vitro (see
Fig. S4 in the supplementary material). CEH-36 binding occurs in parallel and
independent of the binding of CHE-1 to the neighboring ASE motif (see Fig. S4
in the supplementary material). Of the other ASEL-specific gcy genes
(gcy-6,-14,-20), or any other ASEL-specific regulatory factor
(lim-6, lsy-6), only the regulatory region of gcy-14
contains a TAATCC motif in its functionally relevant regulatory region
(lsy-6 also contains such a motif but it is not required for its
correct expression; data not shown), thereby corroborating the overall theme
that the mechanisms of L/R asymmetric regulatory control are diverse.
The contribution of ASE motif affinity to bilateral versus asymmetric ASE motif activity
Apart from cis-regulatory motifs that collaborate with the ASE motif, we
asked whether intrinsic features of an ASE motif may also bear relevance to
its bilateral versus L/R asymmetric activation. Specifically, we wondered
whether the ASE motifs of asymmetrically expressed genes have a weaker
affinity for CHE-1 compared with the affinity of bilaterally expressed ASE
motifs, which would allow co-activating and co-repressing cis-regulatory
mechanisms to modulate the activity of the ASE motifs of asymmetrically
expressed genes. This hypothesis was prompted by the creation of a metric, the
`ASE motif score', which measures the quality of a match of a given ASE motif
to the ASE motif consensus sequence
(Etchberger et al., 2007).
According to this metric, the asymmetrically expressed gcy-5 gene has
a lower motif score than the bilaterally expressed ceh-36 gene (0.63
versus 0.72 in a scale from 0.56 to 0.73)
(Etchberger et al., 2007). To
put the relevance of this metric to an experimental test, we performed
electrophoretic mobility shift assays and found that bacterially produced
CHE-1 indeed binds the ASE motif from ceh-36 with higher affinity
than the ASE motif from the gcy-5 locus
(Fig. 5A,D). The basis for the
difference in motif score and binding affinity appears to be a differential
contact of the respective ASE motifs by CHE-1. Mutating individual zinc
fingers and testing mutated proteins in gel shift assays demonstrates that
CHE-1 contacts the ceh-36 ASE motif with three out of its four zinc
fingers, whereas it contacts the gcy-5 ASE motif only with two out of
four zinc fingers (Fig.
5A).
The low-affinity binding site in gcy-5 may be required to enable the negative regulatory elements (`co-repressor motifs') described above to counteract bilateral ASE motif activation. This hypothesis makes the prediction that if the low affinity ASE motif in the gcy-5 locus were switched with a high affinity ASE motif from the ceh-36 locus, the gcy-5 promoter may become active in both ASEL and ASER. We indeed find that a mere substitution of 4 bp, which transforms the gcy-5 ASE motif into that of the ceh-36 ASE motif, causes de-repression in ASEL (Fig. 5B). To test whether this logic also applies to an ASEL-specific gene, we analyzed the gcy-14 promoter, which also contains an ASE motif with a motif score lower than that of ceh-36 (0.69 versus 0.72). We experimentally confirmed the lower affinity of the gcy-14 ASE motif compared with that of the ceh-36 ASE motif using competitive gel shift assays (Fig. 5D). As is the case for gcy-5, swapping the gcy-14 ASE motif with that of ceh-36 within the context of the gcy-14 promoter also results in bilateral expression of the reporter (Fig. 5B).
Although the logic of asymmetrically expressed genes harboring low affinity ASE motifs appears to apply to some cases, it does not apply generally. The asymmetrically expressed lsy-6 gene also contains a low-affinity ASE motif, as corroborated by competitive gel shift assays, but the two L/R asymmetrically expressed gcy genes gcy-7 and gcy-20 do not (Fig. 5D). Moreover, swapping the ASE motifs of the L/R asymmetric gcy-7 and gcy-22 genes with that of the bilateral, high-affinity ceh-36 ASE motif does not cause de-repression in the contralateral neuron (data not shown). These findings further underscore the diversity of the cis-regulatory mechanisms that control L/R asymmetric gene expression.
An unusual class of che-1 alleles separates the role of che-1 in bilateral fate specification versus asymmetric subtype specification
Further insights into the importance of CHE-1 DNA-binding affinity with
regard to the cis-regulatory logic of left/right asymmetric subtype
specification were obtained by the recovery of an unusual class of
che-1 alleles. In a previously described screen for mutants that
affect ASE specification, we described a mutant locus, lsy-14, that
resulted in the conversion of ASEL fate to ASER fate, exemplified by ectopic
expression of the ASER fate marker gcy-5 in ASEL and a concomitant
loss of the ASEL fate marker lim-6
(Sarin et al., 2007)
(Fig. 6A). This mutant
phenotype is a characteristic phenotype of gene regulatory factors that act
within the gene regulatory loop described in
Fig. 1A. For example,
lsy-6 miRNA mutants cause a similar `2 ASER' phenotype as
lsy-14 mutants (Johnston and
Hobert, 2003).
We mapped the lsy-14(ot101) locus to a small interval on
chromosome I and obtained rescue of the mutant phenotype with a single fosmid
(Table 1), which contains
several genes, including che-1. A genomic fragment containing only
the che-1 locus also rescues ot101
(Table 1). Moreover,
ot101 fails to complement the che-1 null allele
ot66 (Table 1). We
sequenced the che-1 locus in ot101 mutants and found a
mutation in the linker region between zinc finger 1 and 2
(Fig. 6B). The identification
of ot101 as a che-1 allele is an unexpected result as
che-1 null alleles cause a `no ASEL/R' phenotype (class III
phenotype), rather than a `2 ASER' (class II) phenotype
(Chang et al., 2003). We then
revisited some previously described che-1 alleles, all of which had
previously only been analyzed by expression of the lim-6 ASEL fate
marker, which is lost in all available che-1 alleles
(Sarin et al., 2007). We find
that in three additional che-1 alleles, the loss of
lim-6::gfp is not a reflection of overall ASE fate loss as previously
assumed, but is rather accompanied by de-repression of ASER fate
(Table 1). All che-1
alleles that cause the `2 ASER'/class II phenotype cluster around the second
zinc finger (Fig. 6B).
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Class II che-1 alleles result in reduced DNA binding affinity
To understand the phenotype of class II che-1 alleles in more
detail, we examined the effect of these mutations on protein function.
Notably, in contrast to strong loss of function or null alleles, which are
either early nonsense mutations or reside in zinc finger 3 and 4
(Sarin et al., 2007), all
class II alleles relate somehow to the second zinc finger of CHE-1. In
ot124, ot153 and ot223, the structural features of the
second zinc finger are affected, including a residue that is directly involved
in DNA binding (Fig. 6B). In
ot101, the linker between the first and second zinc fingers is
affected (Fig. 6B). Linker
regions have previously been shown to be essential for DNA binding of adjacent
zinc fingers (Choo and Klug,
1993). We bacterially produced CHE-1 proteins that harbor the
ot124 and ot223 point mutations in the second zinc finger.
We find that each protein displays a decreased DNA-binding affinity across
five different tested ASE motifs, both of high and low affinity
(Fig. 6C). Even though it is
unclear how mutations in zinc finger 2 interfere with overall DNA binding of
CHE-1, we conclude that a reduction in DNA-binding activity causes a
disruption of L/R asymmetrically expressed genes (hence, causing a class II
Lsy phenotype), but leaving bilaterally expressed cis-regulatory
control regions unaffected. This underscores the importance of DNA-binding
affinity for controlling L/R asymmetric gene expression programs in the ASE
neurons.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Maintenance of neuronal fate
The amenability of C. elegans to RNAi and the possibility to time
its delivery allowed us to ask the fundamental question whether gene
regulatory factors that turn on a specific terminal neuronal fate also are
continuously required to maintain this fate throughout the life of the neuron.
We find this to indeed be the case for the che-1 transcription
factor. Using a temperature-sensitive allele, such sustained function could
also be demonstrated for the unc-4 homeobox gene, which acts to
determine specific synaptic inputs (Miller
et al., 1992), but to our knowledge this issue has not been
addressed for other regulatory factors that control neuronal differentiation
in any system. Consistent with a maintenance function, che-1 is
expressed in the ASE neurons throughout the life of the animal (data not
shown). Sustained che-1 activity is ensured by che-1
autoregulating its own expression via an ASE motif
(Etchberger et al., 2007).
The continuous expression and requirement for che-1 suggests that
CHE-1 does not only have a role in initiating the terminal fate of ASE
neurons, which is, immediately after the birth of the ASE neurons, initially
bilaterally symmetric (Johnston et al.,
2005). Rather, che-1 also appears to be required for the
progression of the hybrid precursor state to the L/R asymmetric terminal
state. We infer this not only from the continuous expression and requirement
of che-1, but also from the presence of functionally required ASE
motifs in the cis-regulatory regions of L/R asymmetrically expressed genes. We
can exclude the possibility that CHE-1 acts first to initiate L/R asymmetric
genes via the ASE motif, and then to only indirectly restrict expression of
L/R asymmetric genes in either ASEL or ASER, via the activation of
intermediary transcription factors. If this were the case, our promoter
analysis would have identified cis-regulatory motifs that can instruct ASE
expression independently of the ASE motif. Even though we have found motifs
that are required for activation of L/R asymmetric genes, these motifs only
act in conjunction with the ASE motif. Moreover, the elimination of repressor
motifs `bilateralizes' the expression of normally L/R asymmetric genes, again
arguing that bilateral CHE-1 is continually able to drive gene expression in
both ASEL and ASER, but is prevented from doing so in a target gene-dependent
manner.
Restriction of CHE-1 activity drives neuron subtype diversification
CHE-1 acts as a terminal selector gene that determines overall ASE fate by
directly activating the expression of a large battery of bilaterally expressed
genes (Etchberger et al., 2007;
Hobert, 2008). In addition to
this neuron class specification function of CHE-1, we have uncovered several
cis-regulatory mechanisms that restrict CHE-1 activity on several target gene
promoters to either the left or right ASE neuron, thereby driving a subtype
specification program that diversifies these two cellular subtypes from one
another (Fig. 7). The
cis-regulatory mechanisms that restrict CHE-1 activity are promoter dependent
and remarkably diverse. In three promoters (lim-6, gcy-7, lsy-6), we
have found evidence for the existence of three distinct unrelated activator
elements with which CHE-1 cooperates to promote L/R asymmetric expression.
Notably, these activator elements are only required in the context of the
complete regulatory module, as the ASE motif alone can drive bilateral ASE
expression when in complete isolation, but is apparently not able to do so if
in the context of the whole regulatory element. In such context, a cooperating
activator motif is required for ASE expression (K50 binding site in
gcy-7 and bHLH binding site in lsy-6). It is possible that
bilaterally expressed ASE motifs also require such positively cooperating
factors.
Regulation of the gcy-7 and lsy-6 promoters, even though relying on distinct motifs, appears to share a similar logic. CHE-1 appears to be engaged in a tug-of-war with repressor elements in the promoter, which it can only overcome by the presence of an additional activator motif that cooperates with the ASE motif. The activator motif is only required if the repressor elements are present. If both are removed, CHE-1 exerts bilateral control over the promoter in both ASEL and ASER.
By contrast, no discrete repressor motifs were found in lim-6 and
no discrete co-activator motifs were found in gcy-5. It appears
striking that none of the four promoters analyzed here uses similar
strategies, as neither of the identified activator or repressor motifs show
any similarity to one another. Moreover, even though some repressor motifs
found to be required in one promoter (gcy-7) are present in other
promoters (gcy-14, gcy-20), these motifs are not required in these
other cases (data not shown). These findings may indicate an independent
evolutionary recruitment of the many L/R asymmetrically expressed gcy
genes into the regulatory network that controls L/R asymmetry of these
neurons. This diversity is consistent with differences in the expression of
gcy genes in different nematode species
(Ortiz et al., 2006). This
plasticity in the composition of the L/R asymmetric terminal features may
relate to the sensory function of ASE neurons that may need to adapt to
distinct environmental cues in a species-specific manner.
Previous genetic analysis has revealed several candidates for activator and
repressor factors that may act through the cis-regulatory elements that we
have described here to restrict CHE-1 activity
(Fig. 1A). These include the
zinc-finger transcription factor DIE-1 [genetically required to activate
ASEL-expressed genes and repress ASER-expressed genes
(Chang et al., 2004)], the LIM
homeodomain protein LIM-6 [required to repress gcy-5 in ASEL
(Hobert et al., 1999),
Fig. 1A], the zinc-finger
protein FOZI-1 [required to repress gcy-7 in ASER
(Johnston et al., 2006),
Fig. 1A], and two other, as yet
uncloned lsy genes with similar phenotypes to lim-6
[lsy-20 and lsy-26
(Sarin et al., 2007)].
DNA-binding sites are not known for DIE-1, LIM-6 or FOZI-1, and in vitro
gelshift assays have not detected binding of these factors to ASEL or
ASER-specific promoters (data not shown). However, the putative
K50-homeodomain binding site identified in the gcy-7 promoter is a
likely binding site for the CEH-36/Otx homeodomain protein. CEH-36 binds to
the motif in vitro and gcy-7 fails to be activated in ceh-36
mutants. The asymmetric, ASEL-fate inducing activity of CEH-36 is also evident
at the cis-regulatory level as the CEH-36 binding site will convert a
bilaterally expressed ASE motif into a regulatory motif that is more strongly
expressed in ASEL than in ASER (ASE-gcy-7#2 construct in
Fig. 4). Therefore, CEH-36
activity must be somehow lateralized, even though CEH-36 is expressed in both
ASEL and ASER.
Another factor that contributes to the restriction of CHE-1 activity is the affinity of the CHE-1/ASE motif interaction, which we again find to be important for some, but not all, promoters. The affinity argument stems largely from three observations. First, swapping a high-affinity ASE motif from a bilateral promoter into that of two different asymmetric promoters results in the partial bilateralization of promoter activity. We interpret this to mean that, at least in some cases, CHE-1 binding to the ASE motif is weak so as to make it susceptible to the effect of repressor motifs. Increases in the affinity of the CHE-1-binding site counteract the repressive effect. Second, increases in CHE-1 expression disrupt L/R asymmetric promoter activity. Third, we have identified an unusual class of alleles of che-1, which separates the activity of che-1 on bilaterally expressed versus L/R asymmetrically expressed promoters. These alleles cause a general decrease in ASE motif affinity and lead to a disruption of L/R asymmetric gene expression, while leaving bilateral expression intact. We note, however, that the importance of affinity may not be a general theme as not all motif swaps yielded the same results and the effect of the unusual che-1 alleles does not extend to every single L/R asymmetrically expressed gene.
|
),
it will be intriguing to investigate how the regulatory elements defined here
relate to Pol II function.
Feed-forward loops may be a general component of cellular subtype specification programs
The findings described here provide insights into how cellular fates become
progressively restricted during development. Transcriptional regulators often
define broad domains of gene expression that become further restricted,
refined and diversified through added layers of regulatory control. In the
context of terminal neuronal differentiation, an important class of regulatory
proteins are what we term `terminal selector genes'
(Hobert, 2008). These encode
for transcription factors that determine the terminal identity of individual
neuron types by directly controlling the expression of terminal gene
batteries. CHE-1 is such a terminal selector gene, which directly controls the
expression of a large battery of cell fate markers that are shared by ASEL and
ASER (Etchberger et al., 2007).
Other examples for terminal selector genes can not only be found in C.
elegans but also in vertebrates, particularly in the brain
(Hobert, 2008). Although
terminal selector genes define the properties of individual neuron classes,
ensuing subtype specification events further diversify neuron classes, as is
the case in the diversification of the ASEL and ASER subtypes.
Together with our previous analysis of gene regulatory factors in ASE, the
data presented here demonstrates that terminal selector genes participate
directly in the subtype diversification that follows neuron type
specification. The way that CHE-1 achieves this feat may reveal a common theme
in gene regulatory networks that serve to diversify gene expression programs.
CHE-1 interacts with other regulatory modules in a feed-forward loop (FFL)
motif configuration. A conventional FFL consists of a transcription factor A,
controlling factor B, and factor A and B controlling together a target C
(Alon, 2007). Such simple FFL
motifs can have specific properties such as persistence detection or response
acceleration. CHE-1 acts in a more complicated version of the FFL. Besides
activating a single transcription factor (CEH-36) with which it collaborates
to regulate the expression of a target gene (gcy-7), CHE-1 also
activates multiple components of the bistable regulatory loop shown in
Fig. 1A; the regulatory loop
provides a net activity output that then cooperates with CHE-1 in a
promoter-specific manner on a given target gene
(Fig. 7). For some genes, the
loop provides a positive output with which CHE-1 needs to interact to be able
to turn on a target gene (e.g. lim-6 in ASEL). For other target
genes, the loop provides a negative output (e.g. in the form of the LIM-6)
that restricts the ability of CHE-1 to turn on a target gene (e.g. prevents
the activation of gcy-5 in ASEL).
Apart from whatever precise kinetic properties such network motif
configuration may convey, one may view such network architecture as being
reflective of the evolution of gene regulatory networks. The two ASE neurons
may have initially been identical, with CHE-1 controlling the exact same set
of genes in ASEL and ASER. This ancestral state may still be reflected
ontogenetically in the hybrid precursor state through which CHE-1 passes after
the ASE neurons are born. As a segregation of certain features (such as
chemoreceptors) into distinct cells (i.e. ASEL and ASER) can convey
beneficiary selective advantages to an animal [increases in discriminatory
properties (Pierce-Shimomura et al.,
2001; Suzuki et al.,
2008)], additional regulatory mechanisms may have been implemented
downstream of CHE-1 to restrict CHE-1 activity to a subset of target genes
(Fig. 7).
Even though not dissected to the same extent as the ASE system, FFL-loop
dependent subtype specification mechanisms also occur in other systems, such
as the vertebrate retina (Hsiau et al.,
2007) or in the fly ventral nerve cord
(Baumgardt et al., 2007), and
may provide a commonly used regulatory logic for subtype specification.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/1/147/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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