Molecular Biology and Evolution 18:1882-1891 (2001)
© 2001 Society for Molecular Biology and Evolution
Comparative Genomics and Regulatory Evolution: Conservation and Function of the Chs and Apetala3 Promoters
Department of Botany, University of Agricultural Science, Vienna, Austria;
Department of Biochemistry, Max-Planck-Institute for Plant Breeding, Cologne, Germany;
Department of Genetics and Evolution, Max-Planck-Institute for Chemical Ecology, Jena, Germany
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses, indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action and gene regulation.
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Improved understanding of gene regulation is essential for genomics and evolutionary biology. Within species, regulatory pathways control development and organismal responses to environment and may contribute to genetic variation for quantitative traits (Wang et al. 1999
) and disease (Horikawa et al. 2000
; Maleck et al. 2000
). Among species, changes in gene regulation may be fundamentally important for interspecific differentiation (Ting et al. 1998
; Kopp, Duncan, and Carroll 2000
). However, gene regulation is poorly understood, especially in plants.
With the completion of Arabidopsis genomic sequencing and the new 2010 project to determine the function of all Arabidopsis genes, there will be many opportunities to understand gene regulation and its variation within and among species. Large-scale genome annotation is routinely done automatically using database searches and gene prediction programs (Lin et al. 1999
; Mayer et al. 1999
). The latter are usually designed to detect protein-coding regions, leaving the untranslated regulatory sequences of a gene largely unexplored. It is well known that functionally important homologous regions in coding sequences tend to be highly conserved between sibling species. Similarly, it is expected that regulatory elements in untranslated regions might be detected by sequence comparison ("phylogenetic footprinting"; Wasserman et al. 2000
). Here, we test the usefulness of this comparative strategy for the study of gene regulation using two genes, chalcone synthase (Chs) and Apetela3, from 22 cruciferous species.
Recent studies have established a well-supported overview of evolutionary relationships within the Brassicaceae (O'Kane and Al-Shehbaz 1997
; Galloway, Malmberg, and Price 1998
; Koch, Bishop, and Mitchell-Olds 1999
; Koch, Haubold, and Mitchell-Olds 2000, 2001
). Divergence times among various taxa have been estimated using several independent loci and fossil calibrations (Yang et al. 1999
; Koch, Haubold, and Mitchell-Olds 2000
). Consequently, these studies provide a basis for selecting informative species for comparative analysis and provide a key to the understanding of rates and modes of regulatory evolution in the Brassicaceae. Arabidopsis thaliana and many closely related species are diploids with relatively few recent gene duplications. This makes it comparatively easy to identify orthologous loci for comparative studies. However, complications may arise in ancient polyploids such as Brassica (Kowalski et al. 1994
; Lagercrantz 1998
).
To obtain new insights into regulatory evolution, we chose the well-characterized promoter regions of the Chs and Apetala3 genes from selected species of the Brassicaceae. Chalcone synthase participates in plant secondary metabolism and catalyzes the key reaction in flavonoid biosynthesis (Hahlbrock and Scheel 1989
). Although many plant species contain multiple Chs genes (Ryder et al. 1987
; Koes, Spelt, and van der Elzen 1989
; Wingender et al. 1989
; An et al. 1993
; Junghans, Dalkin, and Dixon 1993
; Durbin et al. 1995
; Howles, Arioli, and Weinman 1995
), Chs is single-copy in A. thaliana (Burbulis, Iacobucci, and Shirley 1996
) and most related diploids (Koch, Haubold, and Mitchell-Olds 2000
). The Chs promoter is responsive to UV light, and it has been characterized experimentally in A. thaliana (Hartmann et al. 1998
) and Sinapis alba (Kaiser and Batschauer 1995
; Kaiser et al. 1995
). In addition, detailed information on the cis-acting elements and potential corresponding trans-acting factors of the Chs promoter region are available for many other plant species (Faktor et al. 1997a
; Feldbrügge et al. 1997
; Seki et al. 1997
and references therein).
The floral homeotic gene Apetala3 is a well-known MADS-box regulatory gene responsible for floral organ identity. The function of this gene has been described (e.g., Okamoto et al. 1994
; Yi and Jack 1998
), and its promoter has been analyzed (Hill et al. 1998
). The molecular evolution of the MADS-box genes including Apetala3 has been studied in detail in higher plants (e.g., Purugganan et al. 1995
; Kramer, Dorit, and Irish 1998
; Lawton-Rauh, Alvarez-Buylla, and Purugganan 2000
; Purugganan 2000), and phylogenetic data on Apetala3 from various Brassicaceae are available (Lawton-Rauh, Buckler, and Purugganan 1999
).
In this paper, we address three main questions: (1) Can comparative analysis of promoter sequences among related taxa identify conserved cis-acting regulatory elements? (2) Do conserved promoter elements confer functional patterns of gene regulation? (3) Do patterns of promoter evolution mirror known phylogenetic relationships inferred from nuclear genes, plastidic loci, and noncoding spacer regions of nuclear ribosomal DNA?
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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A set of species from the Brassicaceae was selected on the basis of previous phylogenetic relationships derived from different molecular markers. The various markers analyzed were the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (Koch, Bishop, and Mitchell-Olds 1999
), the nuclear genes Chs and ADH (Koch, Haubold, and Mitchell-Olds 2000
), and the plastidic matK gene (Koch, Haubold, and Mitchell-Olds 2001
). Details on the species and accessions are given in table 1
. We investigated one individual per accession. Alignments of Apetala3 and Chs promoter regions (supplementary material, figs. 1 and 2) are available via the online version of the manuscript.
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DNA Amplification, Cloning, and Sequencing
Total DNA was obtained from leaf tissue of single individuals by a modified CTAB procedure (Mummenhoff and Koch 1994
). Polymerase chain reaction (PCR) was carried out using an ABI 9700 (Applied Biosystems). The PCR cycling scheme was 5 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 50–55°C (depending on primer combination), and 1 min at 72°C; 15 min extension at 72°C; and a final hold at 4°C. The oligonucleotide sequences used to amplify promoter fragments were as follows: Chs—CHS1.for (5'-gagttaagtatgcacgtg-3') and CHS.rev (5'-gagatcagaaggcacagag-3'); Apetala3—APET1.for (5'-ggcttttaacaccaatataaaaa-3'), APET2.for (5'-caatataaaaacttggttcacac-3'), APET3.for (5'-gccaaccaaatccacctgca-3'), and APET.rev (5'-gagagggaagatccagatcaagagg-3'). We designed several forward primers by comparing database sequences from Brassica oleracea (Hill et al. 1998
; AF043610) and A. thaliana (Irish and Yamamoto 1995
; U30729), because DNAs from several taxa did not amplify well with APET1.for. The primer APET2.for overlapped with APET1.for at its 5' end. DNAs which did not provide good PCR results for primer APET1.for or APET2.for originated from Cochlearia pyrenaica, Arabis alpina, Matthiola incana, Arabis glabra, and Arabis turrita (table 1
and fig. 2, supplementary material). DNA extracted from these individuals was amplified using a third primer, APET3.for. This primer is located 40 bp downstream of the APET2.for primer sequence in A. thaliana and 485 bp downstream of the APET2.for primer sequence in B. oleracea. For an overview of the success of different primer combinations in amplifying Apetala3 promoter regions, see table 2
.
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PCR reactions (50 µl) were performed under the following conditions: 50 ng template DNA, 2 ng/ primer, 2.5 mM MgCl2, and 2 U Taq DNA polymerase. All PCR products were purified from an agarose gel using the Boehringer PCR product purification kit and cloned either into pGEM-T cloning vector (Promega) or into the TA kit pCR II cloning vector (Invitrogen). The PCR products from all different Apetala3 amplifications (table 2 ) were cloned as well. For each DNA sample, we performed two independent PCR reactions. From each PCR reaction, two independently cloned PCR products were sequenced separately to detect possible sequence variation due to polymerase errors.
For each promoter fragment, both strands were cycle-sequenced using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (ABI Applied Biosystem, Inc.). Products of the cycle sequencing reactions were run on an ABI 377XL automated sequencer (ABI Applied Biosystem, Inc.). Cloned PCR products were sequenced using universal t7 forward (5'-gtaacgatttaggtgacactatcg-3') and M13-48 reverse (5'-agcggataacaatttcacacagga-3') primers.
Subcloning of Chs Promoter Fragments and Expression Analysis
Based on the Chs promoter sequences, primers were designed which annealed to the ATG start codon region and a position at about -220 relative to the transcriptional start site. The upstream primer added a HindIII site, and the downstream primer added a NcoI site which was placed on the ATG. PCR fragments representing the various Chs promoter sequences were cut with HindIII and NcoI and cloned into similarly digested pBT10-GUS (Sprenger-Haussels and Weisshaar 2000
). Identities of clones were confirmed by sequencing. Expression analysis was performed as described (Hartmann et al. 1998
; Sprenger-Haussels and Weisshaar 2000
) using a luciferase expression construct as an internal control.
Phylogenetic Analysis
The sequence from S. alba was published by Batschauer, Ehmann, and Schafer (1991)
under accession number X17437. Promoter regions were aligned by hand, and highly conserved regions served as anchor regions for successive alignment. Only those parts of the alignment which we judged to be highly reliable were used for phylogenetic analysis. These parts showed pairwise similarities >50% and were not disrupted by extensive microsatellite sequences. We excluded DNA regions which were used as primer-binding sites for amplification. Phylogenetic distances were computed using Kimura's (1980)
two-parameter model, and the resulting distance matrices were subjected to the neighbor-joining algorithm as implemented in TREECON (version 1b; Van de Peer and De Wachter 1997
), which was also used to perform bootstrap analysis (Felsenstein 1985
) with 1,000 replicates.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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DNA Sequence Variation in the Chs Promoter Regions
The Chs promoter fragments we obtained varied in length from 392 bp in Cochlearia pyrenaica to 1,111 bp in Arabidopsis griffithiana (table 1 ). No allelic variation was detected within species. The mean G/C content was 34.9% (SD = 3.1%), with a minimum of 28.5% in the long A. griffithiana promoter fragment. The highest G/C content was found in the short promoter fragment of Fourraea alpina, with 39.4% (table 1 ). The alignment of the entire promoter region is shown in figure 1 . At alignment position 121, we removed from consideration DNA sequences which were not alignable. These fragments were mostly shorter than 75 bp, but in the case of A. griffithiana (657 bp) and S. alba (298 bp), they were much longer.
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In our earlier investigation of Chs coding sequences, we used a forward primer located in the ACE-MRE region (CHS-FOR1 in Koch, Haubold, and Mitchell-Olds 2000
) at alignment position 496 (fig. 1
, supplementary material). DNA sequence comparisons with these data demonstrated that our promoter fragments corresponded to the previously isolated Chs genes, which have been shown to be orthologous (Koch, Haubold, and Mitchell-Olds 2000
). Therefore, our corresponding promoter fragments represented exclusively orthologous DNA regions.
DNA Sequence Variation of Apetala3 Promoter Regions
The sequencing of different subfragments of the Apetala3 promoter region (table 2
) mostly resulted in overlapping fragments of identical sequences. In the case of Rorippa amphibia with the primer combination APET3.for/APET.rev, one additional sequence was found. In Arabis hirsuta, we detected three different genes corresponding to the three primer combinations. However, A. hirsuta is an autotetraploid plant, so we probably isolated alleles from the two duplicated loci.
The alignment of the entire promoter region is shown in figure 2 (supplementary material). The length of the fragment (APET1.for/APET.rev) varied from 486 bp in R. amphibia to 1,036 bp in B. oleracea. The mean G/C content was 34.7% (SD = 1.3%), with a minimum of 32.2% in the C. pyrenaica promoter fragment. The highest G/C content was found in the promoter fragment of A. glabra, with 37.6% (table 1 ). At alignment position 62, we found a 459-bp indel in the B. oleracea promoter.
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Conserved Elements in the Chs Promoter Region
Several conserved elements and regions could be detected among the different Chs promoters (fig. 1 , supplemetary material). A G-box motif described previously by Hartmann et al. (1998)
was located at alignment position 13. Because this motif was part of the amplification primer APET3.for, we were not able to detect mutations at these sites. Amplification worked in all taxa, and hence this element is presumably ubiquitous. An additional G-box was located at position 270. This G-box was only present in the promoters of Arabis jaquinii and Arabis alpina, which form a monophyletic group (Koch, Bishop, and Mitchell-Olds 1999
; Koch, Haubold, and Mitchell-Olds 2000, 2001
). The third G-box was located at position 507, in close proximity to the TATA box (position 604). It has previously been described as a highly conserved promoter region in crucifers, Phaseolus vulgaris (Faktor et al. 1997b
), Petrosilinum crispum (Schulze-Lefert al. 1989a, 1989b
; Block et al. 1990
), Gentiana triflora (Kobayashi et al. 1998
), and Petunia hybrida (Van der Meer et al. 1990
). This G-box motif is also referred to as the ACGT-containing element (ACE; Hartmann et al. 1998
). In close 3' proximity at position 539 there was an H-box motif (Loake et al. 1992
; Faktor et al. 1997b
), which has also been denoted the Myb recognition element (MRE) motif (Hartmann et al. 1998
). Between the MRE and the ACE elements there was an A-box motif, which was first observed in phenylalanine ammonia-lyase (PAL) in P. crispum (Logemann, Parniske, and Hahlbrock 1995
).
Another H-box-like (or MRE) motif was located at position 397. Database comparisons with PLACE, a collection of promoter elements (http://www.dna.affrc.go.jp/htdocs/PLACE/signalscan.html; Higo et al. 1999
) revealed several other conserved cis-acting regulatory elements with unknown functions in the Chs promoter region. There was an A-box motif (TACGTA) at position 220, related to the G-box or MRE with a core ACGT sequence. However, this ACGT sequence only appeared in the distantly related R. amphibia and Cochlearia excelsa. A CAAT promoter consensus sequence has been found at positions 375 and 407. The sequence is highly conserved and might be responsible for tissue-specific promoter activity (Shirsat et al. 1989
).
A consensus GT-1-binding site (GAAAAA) was found at position 452. This motif is present in many light-regulated genes from numerous species (Villain, Mache, and Zhou 1996
; Zhou 1999
). A related GGTTAAA/TA/T motif has been described by Lawton et al. (1991)
in a silencer region of Chs from the bean. Several other highly conserved regions (>50% invariable nucleotide positions in a fragment a minimum of 15 bp in length) have no known functional role and were labeled as "region 1" and "region 2" (fig. 1
, supplementary material; positions 439–555 and 471–488, covering the GT-1-binding site). These conserved sequence motifs may represent novel regulatory elements.
Conserved Elements in the Apetala3 Promoter Region
Several MADS-domain-containing proteins bind DNA at a CC(A/T)6GG core consensus binding site which has been referred to as the CArG box (Schwarz-Sommer et al. 1992
; Wynne and Treisman 1992
; Huang, Mizukami, and Ma 1993
; Shiraishi, Okada, and Shimura 1993
; Hill et al. 1998
). The Apetala3 promoter of A. thaliana contains three CArG box motifs (Hill et al. 1998
), which may serve as binding sites for one or more MADS-domain-containing proteins (Irish and Yamamoto 1995
). These boxes are highly conserved among the cruciferous plants analyzed in this study and are identified in figure 2
(supplementary material) at positions 428, 476, and 500.
Several other highly conserved regions were located exclusively upstream of the CArG1 motif, and none of them have been characterized experimentally. (As for the analyzed Chs-promoter region, we defined regions as "highly conserved" if more than 50% of a DNA sequence with a minimum length of 15 bp was invariable.) However, strong evidence for functional relevance of promoter regions upstream of the CArG1 motif is provided by experimental analysis of the Apetala3 promoter in A. thaliana (Hill et al. 1998
).
The Apetala3 alignment (fig. 2
, supplementary material) shows four conserved regions, which we designated region 1 (alignment positions 73–133), region 2 (alignment positions 216–230), region 4 (alignment positions 365–381), and duplicated region 3a (alignment positions 328–347) and region 3b (alignment positions 383–402). In each of regions 1, 2, and 4, there was one MYB-binding site (with a consensus motif CNGTTR; Urao et al. 1993
). Only in the highly conserved regions 3a and 3b (90% invariable sites) were we unable to identify known regulatory motifs.
Evidence for additional motifs is ambiguous. At position 78 there was a second putative MYB–homolog-binding site, previously recognized in maize (consensus motif CCWACC; Grotewold et al. 1994
). Overlapping with this myb element was a sequence similar to a region necessary for circadian expression of the tomato Lhc (light harvesting complex) gene (CAANNNNATC; Piechulla, Merforth, and Rudolph 1998), and overlapping with the 3' end of this putative myb element was a G-box-like CACCTG motif. At positions 73 and 243 there were also core sites (AAAG) required for binding of Dof proteins (DNA-binding proteins) in maize, with presumably only one zinc finger (Yanagisawa and Schmidt 1999
). Interestingly, a few base pairs upstream of this AAAG recognition sequence, there was an ACTTTA motif (alignment position 53), which has been shown to act as a Dof–protein-binding site (Baumann et al. 1999
). We also found the same motif at alignment position 421 close to the CArG1 box.
Phylogenetic Analysis
With the exception of Lepidium campestre and M. incana, the evolutionary relationships of these species have been considered in previous studies (Koch, Haubold, and Mitchell-Olds 2000, 2001
). A schematic phylogenetic network based on matK and Chs coding regions considering only taxa analyzed herein is redrawn from Koch, Haubold, and Mitchell-Olds (2001)
in figure 2
for further comparisons. The phylogenetic tree estimated from Chs promoter sequence divergence values (fig. 3
) differs from the combined matK/Chs tree (fig. 2
) only in the relative positions of L. campestre and M. incana. However, as outlined above, there is little statistical support (bootstrap values and decay indices in Koch, Haubold, and Mitchell-Olds 2001
) for the relative positions of Lepidium and Matthiola, and therefore the conflict among these differing topologies is statistically insignificant. This means that Chs promoter sequence evolution corresponds closely with Chs gene coding sequences as well as several other genes and DNA regions. This holds at least for the selected portions of the sequences for which a reliable alignment was found. However, distance analysis of the total alignment resulted in the same tree topology with some extremely long branches. Because it is not possible to calculate simple synonymous or nonsynonymous mutation rates for pairwise comparisons, we used the Kimura (1980)
two-parameter distances to compare Chs promoter regions (alignable regions as indicated in fig. 1
, supplementary material) with the corresponding Chs coding regions (Koch, Haubold, and Mitchell-Olds 2000
). These data showed that within the selected promoter regions the mean substitution rate in the Chs promoter region is 0.58 times as low (SD = 0.18) as the mean synonymous substitution rate in the corresponding coding Chs region.
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The phylogenetic topology from the Apetala3 promoter sequences is nearly identical to that obtained from the Chs promoter region (fig. 4 ). We take this as evidence that we analyzed orthologous promoter fragments of the Apetala3 MADS-box regulatory gene. It has recently been shown that there is a large monophyletic assemblage that includes the major floral homeotic gene groups AGAMOUS, Apetala3, PISTILLATA, and APETALA1/AGL9, and these four MADS-box genes were clearly separated from each other in previous phylogenetic analyses (Purugganan et al. 1995
; Purugganan 1997
). Therefore, it is unlikely that we analyzed promoter fragments not related to the Apetala3 gene. Three Apetala3 coding regions have been reported (Brassica napus AF124814, A. thaliana D21125, and Cardaminopsis petraea AF143380). For these three taxa, the mean substitution rate in the apetala3 promoter regions selected for phylogenetic analysis is 0.59 (SD = 0.17) times as low as the synonymous substitution rate in the corresponding coding regions.
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Chs Promoter Expression Analysis
Functional analysis of the Chs promoter fragments from R. amphibia, F. alpina, A. griffithiana, C. petraea, A. alpina, and Aethionema grandiflora revealed no major differences in UV light response. Absolute expression levels varied at most by a factor of two among taxa (fig. 1 ), and Chs induction varied from 75- to 204-fold. Error bars represent standard deviation from eight transfection experiments. These data show that the conserved sequences detected in the various promoters are functional and that variable sites do not interfere with functionality. Our results indicate that conserved promoter elements do indeed have a regulatory function and that sequence comparisons can be used to infer the type and location of important cis-acting elements in promoter sequences. Without additional information, it is still difficult to relate these elements with the response to potential external or internal stimuli. However, the increasing information from parallel expression analysis (Zhu and Wang 2000
) will soon provide the data required for inferring such functional relationships. |
Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Functional Dissection of the Apetala3 and Chs Promoters
Hill et al. (1998)
reported functional dissection of the Apetala3 promoter region in A. thaliana, and identified regions of promoter sequence conservation between A. thaliana and B. oleracea. However, the results of their Arabidopsis-Brassica sequence comparison differed substantially from our results, which are based on 16 sequences from cruciferous plants. Only a few elements, such as the CArG motif and region 4, were apparent in the comparison of Hill et al. (1998)
. The remaining highly conserved motifs (e.g., regions 1, 2, and 3) were not apparent in that study of only two species. Nevertheless, Hill et al.'s (1998)
experiments clearly demonstrated that a promoter region covering our region 1 (fig. 2
, supplementary material) is required for anther-specific expression. This work indicates that our defined promoter regions 3a, 4, and 3b are required for early (stages 3–5) and for petal-specific expression during floral development, while region 2 is required for expression in the filaments. Hill et al. (1998)
concluded that specific temporal and spatial cis-acting elements exist within the Apetala3 promoter. These predicted elements correspond to the conserved regions we identified in this study.
Hartmann et al. (1998)
showed that a light regulation unit (LRU) containing the ACE and the MRE was sufficient for UV/blue light-regulated expression of Chs. The corresponding LRU from bean Chs15 has been intensively studied (Arias, Dixon, and Lamb 1993
), and it has been shown that the G-box (ACE) and the H-box (MRE) make major contributions to the transcription of the Chs15 promoter in vivo. Combinatorial specificity of these elements has also been found in promoters of genes encoding phenylpropanoid biosynthetic enzymes (Lois et al. 1989
; Leyva et al. 1992
). The corresponding LRU regions in Chs promoters of the French bean (Faktor et al. 1996
) and the petunia (van der Meer et al. 1990
) are also responsible for tissue-specific activity. Finally, additional H-boxes binding MYB classes of transcription factors are present upstream of the LRU unit of bean Chs15 (Arias, Dixon, and Lamb 1993
). This corresponds to our observation of a highly conserved H-box-like sequence at alignment position 396 (fig. 1
, supplementary material).
Recently, Seki et al. (1996)
presented an overview of these elicitor-responsive cis elements and clearly showed similarities among Chs and PAL gene promoters from different species. Interestingly, only a few additional regions have been subjected to functional dissection analysis. Nuclear factors (SBF-1) have been identified in P. vulgaris that bind to SBF-1 boxes in the 5' region of the bean Chs15 gene promoter regulating tissue-specific expression (Hotter et al. 1995
). These SBF-boxes contain a consensus motif identical to the GT-1 recognition motif. We found a highly conserved GT1-like motif (fig. 1
, supplementary material) at alignment position 452 which might have a similar function.
Perspective
We analyzed interspecific patterns of sequence conservation in promoter regions of two genes, Chs and Apetala3. Our results show that known functionally important regulatory elements are conserved among crucifer relatives of Arabidopsis (figs. 1 and 2 , supplementary material). Other, previously unknown, elements were identified which are even more highly conserved than the known motifs which have been identified by functional studies. In addition, we showed that Chs promoter sequences from a broad sample of related species are sufficient for light-regulated gene expression in transient expression assays in Arabidopsis.
Comparative analyses of putative regulatory regions are increasingly important in genomics. Sequences of CFTR, the gene responsible for cystic fibrosis in humans, have been compared in humans, mice, and Fugu (Davidson et al. 2000
; Ellsworth et al. 2000
). Human-mouse comparisons found many intergenic and intron segments with high levels of conservation, providing little ability to identify which regions were functionally important in gene regulation. With greater evolutionary divergence, genomic sequence 5' of CFTR is highly divergent between pufferfish and mammals, except for conserved putative CRE and CAAT box elements. Likewise, human-mouse comparison of the SNCA gene, which may influence development of Alzheimer's and Parkinson's diseases, identified a novel 5' element that regulates normal expression in transient assays (Touchman et al. 2001
). Wasserman et al. (2000)
studied conserved promoter regions from 28 human-mouse pairs of skeletally expressed genes and identified a number of known and inferred enhancer elements.
Comparative analysis of promoter sequences from related species is a powerful tool to identify conserved and putatively functional elements. Rapidly accumulating knowledge about the function and regulation of genes coming from the model plant A. thaliana can be easily transferred to its closest relatives. Wild relatives provide a broad spectrum of naturally occurring genotypes and phenotypes, which are presumably adapted to a range of environments and natural histories. This will permit hypothesis testing about the evolutionary significance of character change and suites of traits. Well-supported phylogenies of numerous Brassicaceae and estimates of divergence times provide an important resource for these studies (Koch, Haubold, and Mitchell-Olds 2000, 2001
). These can be used to compare rates of evolution. In our analysis, the greatest evolutionary divergence was about 45 Myr (for the Chs promoter of A. thaliana compared with A. grandiflora).
Bioinformatic analyses have used three approaches to identify putative regulatory regions within promoters: pattern recognition, phylogenetic footprinting among several species, and within-species comparison of promoters from coregulated genes (Fickett and Wasserman 2000
; Wasserman et al. 2000
). Phylogenetic footprinting is particularly important, because it reduces the search space for subsequent computational analysis. In addition, comparison of promoter sequences from several species at different levels of evolutionary divergence may provide improved identification of putative functionally important sites (e.g., Davidson et al. 2000
). Likewise, expression profiling can identify groups of coregulated genes which may display above-average frequencies of particular regulatory motifs (Maleck et al. 2000
). All three approaches will be useful for understanding gene regulation in A. thaliana.
Despite successful comparisons in our experiments and in other studies, regulatory function may be maintained even in the absence of sequence conservation. For example, the Drosophila melanogaster and Drosophila pseudoobscura promoters of even-skipped mediate conserved patterns of stripe 2 expression during embryo development. In contrast, the functionally important stripe 2 enhancer shows considerable divergence between these two species. Chimeric promoters (containing both combinations of the 5' and 3' halves of the D. melanogaster and D. pseudoobscura upstream regions) are no longer expressed in the wild-type pattern (Ludwig et al. 2000
). This decoupling of conservation and function will complicate attempts to identify functionally important regulatory motifs by comparative genomics.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Sequence alignments are provided on the Molecular Biology and Evolution website.
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank Domenica Schnabelrauch and Antje Figuth for help with the sequence analysis. This work was supported by the Max-Planck-Gesellschaft, and by grants to T.M.-O. from the U.S. National Science Foundation (DEB-9527725) and the European Union.
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Footnotes |
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Julian Adams, Reviewing Editor
1 Keywords: Apetala3
Chs, comparative genomics
phylogeny
promoter function
regulatory evolution 
2 Address for correspondence and reprints: Marcus A. Koch, Department of Botany, University of Agricultural Sciences, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria. koch{at}edv1.boku.ac.at
.
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References |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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