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First published online 14 December 2005
doi: 10.1242/dev.02185
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1 Department of Genetics, Stanford University, Stanford, CA 94305, USA.
2 Stanford Medical Informatics, Stanford University, Stanford, CA 94305,
USA.
3 Department of Developmental Biology, Stanford University, Stanford, CA 94305,
USA.
4 AbGenomics Corporation, Neihu, Taipei 114, Taiwan.
* Author for correspondence (e-mail: kim{at}pmgm2.stanford.edu)
Accepted 26 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: C. elegans, Intestine, Gene expression, GATA transcriptional regulation, Chromosomal clustering
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sulston et al., 1983). Second,
there are exceptionally good genomic resources with which to study development
at the systems level. The complete genome sequence of C. elegans has
been determined, DNA microarrays and an RNAi library containing nearly every
gene in the genome are readily available
(Jiang et al., 2001;
Kamath and Ahringer, 2003),
and protein interactions have been studied on a large scale
(Li et al., 2004). In order to refine our understanding of development from cellular to molecular resolution, our aim is to define most or all of the genes expressed in each of the major tissue types. A global developmental profile of gene expression in C. elegans will elucidate the genes expressed in specific tissues and in all tissues, expand our understanding of tissue differentiation, and lead to insights in regulation of tissue-specific gene expression.
The small size of C. elegans (1 mm in length) makes it impractical
to measure gene expression directly by dissecting tissues. One approach used
to identify genes expressed in a cell lineage or tissue is mRNA tagging
(Roy et al., 2002). Genes
expressed in the body wall muscle were identified by expressing an
epitope-tagged protein that binds poly-A tails on messenger RNA (poly-A
binding protein or PAB-1) from the muscle-specific promoter for the gene
myo-3. Epitope-tagged PAB-1 in the muscle was crosslinked to mRNA in
that tissue, the PAB-1/mRNA complexes were enriched by immunoprecipitation
with an antibody to the epitope, and DNA microarrays were used to identify
1354 muscle-expressed genes (Roy et al.,
2002).
In order to extend tissue profiling in C. elegans, we have
employed mRNA tagging to identify genes expressed in the intestine. C.
elegans is a filter feeder with a digestive system composed of three main
parts: pharynx, intestine and rectum. The pharynx concentrates and processes
food before passing it to the intestine. The intestine is a tube that twists
180° along its length and is composed of twenty epithelial cells with a
layer of microvilli that surround a lumen
(White, 1988). The 14
posterior-most intestinal cells undergo nuclear division at the beginning of
the L1 larval stage and become binucleate. All 20 cells undergo
endoreduplications of their DNA at each larval stage, making the adult
intestinal nuclei 32-ploid (Hedgecock and
White, 1985). The intestine secretes digestive enzymes into the
lumen, absorbs processed nutrients, functions as a storage organ with granules
packed with lipids, proteins or carbohydrates, and nurtures germ cells by
producing yolk proteins that are transported to the oocytes
(Kimble and Sharrock, 1983).
The third part of the digestive tract, the rectum, is composed of endothelial
and muscle cells.
The regulatory network of transcription factors that direct the
differentiation of the intestine has been studied in detail. The P1 cell
differentiates into the EMS and P2 cells by SKN-1-dependent activation of
med-1 and med-2 in the EMS cell and PIE-1-dependent blocking
of SKN-1 in the P2 cell (Maduro et al.,
2001). EMS divides into the E and MS cells. end-1 and
end-3 are the direct targets of MED-1 and MED-2, and are consequently
expressed in the E cell (Maduro et al.,
2001). END-1 and END-3 induce the expression of elt-2 and
elt-7, leading to the activation of downstream targets that
differentiate the E cell into the 20 intestinal cells
(Fukushige et al., 1998;
Maduro and Rothman, 2002).
elt-2 and elt-7 expression is maintained into adulthood by
an autoregulatory loop, propagating intestinal cell identity
(Maduro and Rothman,
2002).
The organogenesis of the C. elegans intestine has been detailed at
the cellular level (Leung et al.,
1999). It includes cytoplasmic polarization of cells in the
intestinal primordium, intercalation of specific sets of cells, generation of
an extracellular cavity within the primordium, and adherens junction
formation. The adherens junctions present an ideal model with which to
investigate epithelial cell polarity and several proteins involved in the
process have been identified such as PAR-3, PAR-6, PKC-3, SMA-1, ERM-1,
LET-413, DLG-1, AJM-1 and others (Knust
and Bossinger, 2002). A molecular profile of the intestine would
help to identify more genes involved in cell polarity and its development.
By generating a profile of gene expression in the C. elegans intestine, we have identified the molecules that define intestinal function. The list of intestine-expressed genes includes genes of known and unknown function. The genes with known functions provide insight into mechanisms and pathways used in diverse intestinal functions, such as epithelial cell polarity, digestion, and resistance to pathogens and toxicity. The intestinal expression of genes with previously unknown function implies a role in intestinal processes.
A genome-wide profile of intestinal gene expression can also be used to elucidate the regulatory networks that maintain intestinal differentiation. We have defined intestine-specific target genes and transcription factors. We searched for DNA sequence motifs enriched in the promoters of the intestine-enriched genes that might function as cis-acting regulatory motifs. This analysis allowed us to generate a first draft of the intestinal regulatory network by linking intestinal transcription factors to their targets via DNA motifs in their promoters.
In addition to identifying muscle-expressed genes, Roy et al.
(Roy et al., 2002) were able
to show that these genes are positionally clustered on the chromosomes. We
have shown that intestine-expressed genes are also located in chromosomal
clusters. Interestingly, we found a strong bias for chromosomal clustering in
the housekeeping rather than the intestine-enriched genes, suggesting a role
of chromatin organization in the regulation of gene expression.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
integrating the plasmid using 
-irradiation (3300 rads) resulting in
strain SD1084.
mRNA tagging
The mRNA-tagging protocol was carried out as described by Roy et al.
(Roy et al., 2002) with
modifications (contact authors for details). RNA was linearly amplified as
previously described (Wang et al.,
2000). The DNA microarrays, probe preparation and microarray
hybridizations were carried out as previously described
(Roy et al., 2002).
Data analysis
All raw data can be found and downloaded from the Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo,
GSE2626) or the Stanford Microarray Database
(http://genome-www.stanford.edu/microarray).
To identify genes that are significantly enriched by mRNA tagging, we first
normalized the total amount of Cy3 and Cy5 signal to each other in each
hybridization. We measured the ratio of the signals from the
co-immunoprecipitated mRNA (Cy5) to total RNA in the cell extract (Cy3), and
calculated the percentile rank for each gene relative to all genes in each
hybridization. The mean percentile rank was determined from eight repeats of
the mRNA-tagging experiment. Student's t-test was used to determine
which genes showed a mean enrichment significantly greater than the median
enrichment for all genes (P<0.001).
To generate tissue-specific and common gene lists, we used the P
values from Student's t-tests used to calculate significant
enrichment in muscle, intestine, and germline. The P values for
muscle-expressed genes were calculated as described by Roy et al.
(Roy et al., 2002) and as
described above for intestine-expressed genes. The P values for germ
line-enriched genes were calculated from a Student's t-test between
log ratios for four repeats of wild-type and glp-4 animals at both
the L4 and adult stages as described by Reinke et al.
(Reinke et al., 2004).
Commonly-expressed genes had a P value of less than 0.05 in the
muscle, intestine and germline DNA microarray experiments. Tissue-enriched
genes had a P value of less than 0.01 in the DNA microarray
experiment involving the tissue of interest and greater than 0.5 in the
experiments concerning the other two tissues. Analysis of chromosomal
clustering was carried out as described by Roy et al.
(Roy et al., 2002).
Motif search
The CompareProspector algorithm was used to search for regulatory motifs as
described by Liu et al. (Liu et al.,
2004). The Pearson correlation coefficient was calculated for
every pair of genes in expression data from 979 C. elegans DNA
microarray experiments (Stuart et al.,
2003).
Construction of GFP reporters
The promoter::GFP constructs for gst-42, elo-6, D2030.5, ZK970.2,
C25E10.8 and B0218.8 were obtained from D. Dupuy
(Dupuy et al., 2004).
Transgenic strains expressing GFP from the promoter of each gene were made by
microinjecting pha-1(e2123) animals with promoter::GFP (50 ng/µg)
and pha-1(+) (pC1, 100 ng/µl, a gift from A. Fire), generating an
extrachromosomal array. The resulting strains for each gene were SD1245 and
SD1246 for D2030.5; SD1144 for elo-6; SD1145, SD1242 and SD1243 for
gst-42; SD1149 for ZK970.2; SD1147 for C25E10.8; and SD1146 for
B0218.8.
Site-directed mutagenesis
One TGATAA site in the promoters of D2030.5, gst-42, and
elo-6 was changed to GGTACC, a KpnI restriction site used as
a diagnostic for mutagenesis, and confirmed by sequencing (contact authors for
details). The mutated promoter::GFP constructs were used to generate
transgenic strains as previously described. Strains with extrachromosomal
arrays expressing GFP from the mutated promoters were generated for
elo-6 (SD1228, SD1229 and SD1230), D2030.5 (SD1159, SD1160 and
SD1161) and gst-42 (SD1162 and SD1163).
GATA transcription factor RNAi
NGM agar with 1 mM IPTG and 25 µg/µL carbenicillin was seeded with
bacteria expressing dsRNA for each targeted gene
(Kamath and Ahringer, 2003),
as well as a negative control with bacteria expressing empty vector. Twenty L4
animals were picked onto each plate, transferred to a fresh RNAi plate after 2
days, and analyzed for GFP expression after 1 day. Four promoter::GFP lines
were treated with RNAi to unc-22 as a negative control and all four
showed no significant change in GFP expression.
Imaging and quantification of GFP expression for mutagenesis and RNAi
Twenty animals for each strain were analyzed for GFP expression using a
Zeiss Axioplan microscope equipped with a CCD camera. Comparison of all images
was carried out on the same day with the same microscope settings. The color
images were converted to 8-bit images using ImageJ software
(Rasband, 2004) and the
measure tool was used to measure pixel intensity of each worm.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To obtain a profile of gene expression in the fully differentiated
intestine, we made extracts from a synchronous population of animals in the
fourth larval stage. We crosslinked polyadenylated mRNA to FLAG::PAB-1, and
enriched for mRNAs expressed in the intestine using anti-FLAG monoclonal
antibodies for immunoprecipitation. Endogenous PAB-1 bound to mRNA in the rest
of the worm does not have the FLAG tag and should not be immunoprecipitated
with the FLAG antibody. The mRNA was extracted from FLAG::PAB-1/mRNA in the
intestinal precipitate and used to prepare cDNA labeled with Cy5. mRNA from
whole worm lysate was isolated from the same extract (before
immunoprecipitation with 
-FLAG antibody) and used to prepare cDNA
labeled with Cy3. These two samples were hybridized to DNA microarrays
representing 
94% of the genes in the C. elegans genome
(Jiang et al., 2001). The
mRNA-tagging experiment was repeated eight times to gain enough statistical
power to distinguish enriched from unenriched mRNAs.
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We performed several controls to verify that the 1938 genes identified by
mRNA tagging truly represent genes expressed in the intestine. First, 1938
enriched genes is much greater than the 18 genes expected by chance (at
P<0.001) out of 18,345 genes on the DNA microarrays. Second, we
generated a list from published literature of 80 genes that are not expressed
in the intestine and showed that only one (1.25%) is in the list of 1938 genes
(see Table S2 in the supplementary material). Third, the intestine gene list
contains 271 genes whose expression pattern has been previously studied (see
Table S3 in the supplementary material). Of these, 190 (70%) are expressed in
the intestine. For many of the remaining 84 genes (30%), previous studies may
have focused on expression in specific cells or tissues and may not have
scored expression in the intestine. The fraction of genes from the list of
1938 mRNA tagged genes expressed in the intestine (at least 70%) is much
higher than the fraction expressed in the intestine from a random set of
genes. When 51 genes are chosen at random, only 13 (25%) are expressed in the
intestine (Roy et al.,
2002).
We analyzed the anatomical expression of six of the 1938 intestine-enriched
genes (elo-6, gst-42, D2030.5, ZK970.2, C25E10.8 and B0218.8) by
observing expression of GFP reporters (Fig.
3). These genes were selected because their expression profile had
not been reported, they represent a range of enrichment values from the
mRNA-tagging experiment, and DNA constructs containing promoter::GFP reporters
have been made available to the C. elegans research community
(Dupuy et al., 2004).
Pgst-42::GFP and Pelo-6::GFP are
expressed strongly in the intestine with some expression in the pharynx.
PD2030.5::GFP is expressed in the intestine as well as
other tissues. PZK970.2::GFP, PC25E10.8::GFP,
and PB0218.8::GFP are expressed at low levels mostly in
the midgut. Genes expressed solely in the intestine would be expected to have
higher enrichment values from mRNA tagging than genes expressed in the
intestine as well as other tissues. gst-42 and B0218.8 have
enrichment values of 0.92 and 0.97, respectively, and show intestine-specific
expression. By contrast, D2030.5 has a lower enrichment value of 0.68 and is
expressed in the intestine as well as most other tissues. A promoter::GFP
reporter strain expressing a seventh gene (K11D2.2) in the list of 1938
intestine-expressed genes is also expressed in the intestine (Y.L.,
unpublished). In summary, all seven GFP reporter genes were expressed in the
intestine, indicating that a high fraction of the 1938 genes identified by
mRNA tagging are expressed in this tissue.
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Comparison of gene expression in the intestine, muscle and germline
Genes specific to the intestine define its unique functions, whereas genes
expressed broadly (housekeeping genes) describe the cellular and metabolic
functions common to all tissues. Defining the set of genes specifically
expressed in the intestine is a necessary first step in order to understand
the transcriptional regulatory networks that drive its differentiation. In
addition to intestine-enriched genes, genome-wide profiles of muscle-expressed
and germ line-enriched genes have been previously defined. A list of 1364
genes expressed in the body-wall muscle was discovered using mRNA tagging
(Roy et al., 2002). By
comparing gene expression levels in worms with a germline (wild type) with
worms without a germline (glp-4 mutants) on DNA microarrays
(Reinke et al., 2004), 3144
genes were shown to be enriched in the germline.
We used the lists of intestine, muscle and germline genes to show which are tissue enriched and which are commonly expressed. We defined housekeeping genes as those that were identified in the intestine, muscle and germline with significant enrichment values at P<0.05. This criterion generated a list of 510 genes expressed in these three tissues (see Table S5 in the supplementary material).
In order to characterize the function of the 510 commonly expressed genes,
we compared them with other sets of co-expressed genes in the C.
elegans gene expression topomap (Kim
et al., 2001). The gene expression topomap is an assembly of 553
microarray experiments that can be used to identify genes that are
co-expressed across diverse experimental conditions. Groups of co-expressed
genes are visualized as gene mountains on a two-dimensional scatter plot such
that the distance between two genes indicates the amount of correlation in
expression. The 510 commonly expressed genes are enriched on mountains 2, 7,
11,18, 20 and 23 (Fig. 4B). Previous work has shown that all six of these mountains are enriched for genes
expressed in the germline, indicating a close association between housekeeping
genes and the maternal germline. Housekeeping genes are expressed in the
maternal germline and packaged into embryos in order to allow expression of
new proteins before the start of transcription at the four-cell stage
(Seydoux and Fire, 1994).
We identified tissue-enriched genes by counting genes that have a
P value of less than 0.01 for one tissue, but greater than 0.5 for
the remaining two tissues. Using these criteria, we identified 624
intestine-enriched, 230 muscle-enriched and 1135 germ line-enriched genes (see
Tables S6, S7 and S8 in the supplementary material). We plotted the
tissue-enriched lists on the gene expression topomap and found that the 624
intestine-expressed genes are highly enriched on mountain 8, which was
previously found to be enriched for intestine genes (151 genes, representation
factor 5.8, P<4.3x10-74)
(Kim et al., 2001)
(Fig. 4C). Intestine-enriched
genes are also enriched on mountains 19 and 21, which contain lipid metabolism
genes. This observation is consistent with the role of the intestine as the
fat storage and lipid metabolism organ in C. elegans. The 1135 germ
line-enriched genes overlap with mountains 7 and 11 (enriched for early
germline genes) and the 230 muscle-enriched genes overlap mountain 16
(enriched for muscle genes) and mountain 1 (enriched for neuromuscular genes)
(Fig. 4D,E). Genes expressed in
the muscle and germ line have been discussed in previous work, and are not
discussed further for the sake of brevity
(Reinke et al., 2004;
Roy et al., 2002).
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There are 295 intestine-enriched genes that do not show sequence similarity to genes that have been studied previously. We made use of the gene expression topomap to identify possible functions for these novel genes. Sixty-six genes are in mountain 8 (enriched for intestine genes), further reinforcing their role in the intestine. Intestine-enriched genes are also overrepresented on mountain 19 (26 genes, representation factor 7.4, P<1.38x10-15) and mountain 27 (5 genes, representation factor 3.1, P<0.023). These two gene expression mountains are each enriched for genes known to function in amino acid metabolism, lipid metabolism and energy generation. The 31 intestine-enriched genes in these mountains may also function in these metabolic and energy pathways.
A bias in chromosomal clustering of commonly-expressed versus tissue-enriched genes
Previous work has shown that co-expressed genes cluster on the chromosomes
of yeast, worms, fruit flies, humans, mice and rats
(Boutanaev et al., 2002;
Cohen et al., 2000;
Kruglyak and Tang, 2000;
Lercher et al., 2003;
Lercher et al., 2002;
Roy et al., 2002;
Spellman and Rubin, 2002). One
possibility is that clustering of co-expressed genes could be due to the
influence of chromatin domains on gene expression. This explanation is
compatible with the long-standing hypothesis that open areas of chromatin are
accessible to transcription factors and are therefore areas of active gene
expression. Another possibility is that a single locus control region could
activate a cluster of closely spaced genes.
To determine if the 1938 intestine-expressed genes are physically clustered, we plotted their chromosomal position and counted the number of times there were two or more genes with translation start sites within 10 kilobases (kb) of each other (Table 1). We excluded genes that are in operons or are a result of recent gene duplications because these genes have similar regulatory elements and would be expected to be co-regulated. Fig. 5 shows an example of an intestinal gene cluster composed of eight genes: two that are highly enriched in the intestine (P<0.001), four that are moderately enriched (P<0.01) and two that are not enriched in the intestine. Out of 1746 intestine-expressed genes, 684 have chromosomal positions within 10 kb of each other, which is significantly more than the number that we would expect to see by chance (519 genes) when 1746 genes are sampled randomly from the genome 10,000 times (P<1x10-15). The gene clusters include 291 genes that were not selected in the mRNA-tagging experiment, of which 24 have P-values less than 0.05 and could therefore also be expressed in the intestine. The observation that intestine-expressed genes are clustered in close proximity to each other on the chromosomes confirms and extends previous results showing clustering of genes that are similarly expressed.
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Regulation of intestine gene expression by GATA transcription factors
To uncover transcriptional and regulatory networks that drive gene
expression in the intestine, we looked for genes that encode putative
transcription factors in the list of 1938 intestine-expressed genes. There are
roughly 473 genes that encode putative transcription factors in the genome, of
which 29 are present in our intestine gene list. Sixteen of these genes are
from the nuclear hormone receptor/zinc finger protein family, three genes have
BZIP domains, four have homeodomains, two are GATA transcription factors, two
have DM (dsx and mab-3)-DNA binding domains, one has a
helix-turn-helix DNA binding domain, and one has a domain similar to the
vertebrate transcription factor enhancer protein TEF-1.
Next, we wanted to identify cis-acting regulatory elements that could
control expression of intestine genes. We used CompareProspector
(Liu et al., 2004) (available
at
http://CompareProspector.stanford.edu)
to search for DNA sequence motifs that are over-represented in the promoter
regions of the 1938 intestine-expressed genes. In this search,
CompareProspector started with the 1000 base pairs upstream of the ATG
translation start site and narrowed the search region by selecting sequences
that are conserved between C. elegans and C. briggsae. A
Gibbs sampling algorithm was employed to search for sequences that are
over-represented compared with random DNA sequence.
The top-ranking DNA motif found by CompareProspector was the consensus sequence T/AGATAA/T, which is the binding site for GATA transcription factors (Fig. 6A). The GATA motif is found in 820 out of 1750 intestine-expressed genes, representing a twofold enrichment over the rest of the genes in the genome (see Table S9 in the supplementary material). GATA transcription factors direct the development of the intestine in a regulatory cascade, as detailed in the Introduction of this paper.
If the GATA motif were functional in the intestine of the L4/young adult
worm, then genes with more GATA motifs should be more tightly co-regulated. To
see if this was true, we calculated the average pairwise Pearson correlation
to measure the co-regulation of these genes across 979 C. elegans
microarray experiments (Stuart et al.,
2003). The list of 1938 intestine-expressed genes contains 554
genes with one GATA site, 193 with two GATA sites and 73 with three or more
GATA sites. The average pairwise Pearson correlation increases from 0.11 to
0.13 to 0.17 for genes with one, two and three GATA motifs, respectively,
further indicating that the GATA motif is functional in intestinal gene
expression.
We determined that there is a higher enrichment of genes with GATA sequence sites in the list of 624 intestine-enriched genes (54%) compared with 510 commonly expressed genes (33%) (Fig. 6B). Furthermore, we showed that a higher number of GATA sequence sites per promoter region generally indicate a higher enrichment value in the intestine. As the number of GATA sites increases, the distribution of enrichment in the intestine shifts to higher values, which we previously found was correlated with intestine-specific expression (Fig. 6C). These results suggest that GATA sequence sites are preferentially associated with genes that are expressed specifically in the intestine rather than generally in all cells.
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We also wanted to know which GATA transcription factors are important for
the regulation of intestine expressed genes in trans. There are 11 putative
GATA transcription factors in the C. elegans genome. For seven of
these, we used RNAi to determine whether reducing their activity would affect
expression of six of the GFP reporters with GATA sites. The seven GATA
transcription factor genes include elt-2 and elt-3, which
are in the list of 1938 intestine genes, and were previously known to have
intestinal expression in the adult. end-1 and end-3 are
expressed in the intestinal E lineage in the embryo
(Maduro and Rothman, 2002;
Zhu et al., 1997). egl-18,
elt-1 and elt-6 are not reported to be expressed in the
intestine (Koh and Rothman,
2001; Page et al.,
1997).
Table 2 shows the results of using RNAi on the GATA transcription factor genes for the six GFP intestinal markers. We quantitatively determined the average level of GFP expression of animals growing on bacteria expressing double-stranded RNA for one of the GATA transcription factors and animals growing on bacteria expressing an empty vector. Student's t-test was used to determine if there was a significant reduction in GFP expression (P<0.001). The reduction of elt-2 function by RNAi decreased GFP expression for four intestinal markers. Reducing the function of end-1, end-3 and elt-3 each reduced GFP expression of 1 or 2 genes. Although egl-18, elt-1 and elt-6 do not have reported intestinal expression, RNAi treatment of these genes decreased expression of two to five intestinal markers. This observation could be due to previously undetected expression of these genes in the intestine or to effects on intestinal expression in response to RNAi treatment in other tissues. As a negative control, we showed that RNAi of unc-22 (a gene expressed in the body wall muscle) did not significantly reduce expression of four out of four GFP markers tested (data not shown).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Roy et al., 2002). Gene
expression profiles of the hypodermis and pan-neuronal tissues would complete
the transcriptional identities of the five main tissue types in the adult
hermaphrodite.
However, purifying RNA from specific tissues for gene expression analysis
in C. elegans is not trivial because of its microscopic size. For
this reason, alternate methods have been described to profile tissue-specific
gene expression in the worm. One such method uses FACS sorting to isolate
cells from primary embryonic cell culture that express GFP from a
tissue-specific promoter (Christensen et
al., 2002; Zhang et al.,
2002). mRNA purified from these cells is then profiled on
microarrays (Zhang et al.,
2002). However, this method captures mRNA from tissue culture,
which may differ in expression from the intact organism. A second approach
compares gene expression in wild-type animals with mutants that lack specific
tissues. This method was employed by Reinke et al.
(Reinke et al., 2000;
Reinke et al., 2004) to
compare animals with and without a germline to identify germline-expressed
genes and by Gaudet and Mango (Gaudet and
Mango, 2002) to compare mutant embryos that produced either excess
or no pharyngeal cells to identify candidate pharyngeal genes. However, many
tissues are necessary for the development and survival of the animal (such as
muscle, intestine, and pharynx). Mutants lacking these tissues die before
hatching and thus RNA must be prepared from embryos before development has
been completed.
A third method is mRNA tagging, which was devised by Roy et al.
(Roy et al., 2002) to identify
genes expressed in body wall muscles and used by Kunitomo et al.
(Kunitomo et al., 2005) to
identify genes expressed in ciliated sensory neurons. We used mRNA tagging to
identify genes expressed in the intestine because it allowed us to look at
gene expression in intact organisms. Once we had identified
intestine-expressed genes, we compared them with previously identified
muscle-expressed and germ line-enriched genes. We identified genes that are
commonly expressed between tissues or enriched in one tissue, thus implicating
them in housekeeping versus tissue-specific pathways.
Previous work has shown that genes in close proximity show correlated
expression in yeast, worms, fruit flies, humans, mice and rats
(Boutanaev et al., 2002;
Cohen et al., 2000;
Kruglyak and Tang, 2000;
Lercher et al., 2003;
Lercher et al., 2002;
Roy et al., 2002;
Spellman and Rubin, 2002).
What mechanisms could cause co-expressed genes to be positionally clustered on
chromosomes? One possibility is that chromatin domains cause chromosomal
clustering (Weintraub, 1984).
In any particular tissue or cell, the genome is divided into regions of open
and closed chromatin, corresponding to regions of active or inactive gene
expression. Genes that are expressed in that tissue would be clustered in open
chromatin regions. Another possibility (not exclusive of the first) is that a
single DNA site simultaneously induces the expression of several genes in
close proximity. For example, the globin genes in mammals are located in a
gene cluster, and high levels of expression of these globin genes requires a
single locus controller that affects expression of each gene in the cluster
(Hebbes et al., 1994;
Stalder et al., 1980). In
C. elegans, DAF-12 DNA response elements have been shown to reside
within clusters of DAF-12-regulated genes
(Shostak et al., 2004).
Enhancer elements can act over very large distances and many are located in
regions 3' to a gene (Valarche et
al., 1997). It is possible that many enhancers have effects on
nearby genes in a manner similar to the globin locus control region or
DAF-12-regulated gene clusters.
These mechanisms for chromosomal clustering make distinct predictions about the relative amounts of chromosomal clustering in housekeeping versus tissue-specific genes. The chromatin domain mechanism predicts that housekeeping genes should show a higher level of chromosomal clustering than tissue-specific genes because housekeeping genes are constrained to be in chromatin domains that are open in all tissues (which are just a subset of the open chromatin domains in a particular tissue). The locus controller/enhancer mechanism does not necessarily predict that there would be a difference in chromosomal clustering between housekeeping and tissue-specific genes because enhancers could act over a distance for both sets of genes.
The data in this paper help distinguish between the two mechanisms for
chromosomal clustering. We have shown that genes that are commonly expressed
show more significant clustering than genes that are specific to intestine and
muscle. Similarly, Lercher et al. (Lercher
et al., 2002) found that genes commonly expressed by 14 human
tissues were chromosomally clustered. By contrast, genes that were specific to
those tissues were not clustered. These results support the chromatin domain
mechanism for chromosomal clustering.
To gain further insight into the regulation of intestine-expressed genes,
we searched for over-represented DNA sequence motifs in the promoters of these
genes and found an enrichment of GATA sequence sites. Several GATA
transcription factors are necessary for the development of the intestine.
Specifically, two redundant genes elt-2 and elt-7 are
responsible for maintaining intestinal cell identity
(Fukushige et al., 1998). There
are only a few targets known to be regulated by elt-2, including a
cysteine protease gene (gcp-1)
(Ray and McKerrow, 1992), two
metallothionein genes (mtl-1 and mtl-2)
(Moilanen et al., 1999), and
several vitellogenin genes (vit-2, vit-5 and vit-6)
(MacMorris et al., 1992;
MacMorris et al., 1994;
Spieth et al., 1985;
Spieth et al., 1991;
Zucker-Aprison and Blumenthal,
1989). By generating a molecular profile of the intestine, we have
identified 820 intestine-expressed genes that have GATA sequence sites in
their promoters and may be targets of GATA transcription factors, such as
elt-2 and elt-7. These target genes may maintain intestinal
cell identity in the adult worm and may be involved in intestinal processes
such as cell polarity, secretion, digestion, nourishment of embryos and
defense against pathogens.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/2/287/DC1
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ACKNOWLEDGMENTS |
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