*Human Genetics Center, University of Texas-Houston;
Howard Hughes Medical Institute, University of Michigan Medical Center;
Department of Biology and Biochemistry, University of Houston;
Departments of Zoology and Genetics, Iowa State University;
||Department of Oral Biology, School of Dentistry, Medical College of Georgia;
¶Department of Ecology and Evolution, University of Chicago
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Abstract |
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Introduction |
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Pax genes contain a paired box that encodes a well-conserved DNA-binding domain of 128 amino acids. In mammals, nine Pax genes (denoted Pax-1 to Pax-9) have been identified, all of which have essential roles in embryonic development of tissues and organs. Pax genes have been implicated in human congenital aberrations (Pax-2, 3, 6, 8, 9) and are also associated with oncogenesis (Pax-3, 5, 7) (reviewed in Engelkamp and van Heyningen 1996
; Dahl, Koseki, and Balling 1997
; Underhill 2000
). Pax gene function is required for normal development of many organs and tissues, ranging from the central nervous system (Pax-2, 3, 5, 6, 7, 8) and eye (Pax-2, 6), to pancreas (Pax-4, 6), and B-lymphocytes (Pax-5) (reviewed in Engelkamp and van Heyningen 1996
; Stuart and Gruss 1996
; Dahl, Koseki, and Balling 1997
; Underhill 2000
). Pax genes have also been isolated from Drosophila (Bopp et al. 1986
, 1989
; Baumgartner et al. 1987
; Noll 1993
; Quiring et al. 1994
; Fu and Noll 1997
; Czerny et al. 1999
), cnidarians (Sun et al. 1997
; Gröger et al. 2000
; Miller et al. 2000
), and other animals such as sea urchins and Caenorhabditis elegans (Chisholm and Horvitz 1995
; Czerny et al. 1997
). On the basis of a phylogenetic analysis of paired domain sequences, the known Pax genes were divided into five groups within two supergroups: Pax-2, Pax-5, Pax-8, Pax-B, poxn/Pax-A, and Pax-6/ey in supergroup I; Pax-1/Pax-9/poxm and Pax-3/Pax-7/gsb/gsbn in supergroup II (Sun et al. 1997
). Pax genes in the same group often display similarities in expression pattern, which is suggestive of similar or partly overlapping roles in development (Chalepakis et al. 1993
). The Pax genes arose via duplication, and their functional diversification is caused in part by the divergence of DNA-binding specificity and affinity of their paired domains. During evolution, amino acid changes in the paired domain are required to convert an ancestral DNA-binding property into a novel binding property. However, many neutral or near-neutral amino acid substitutions also accumulate, making it difficult to identify critical amino acid residues. For example, only three of the 30 amino acid differences between Pax-5 and Pax-6 paired domains were found to be important for the differences in DNA-binding specificity between the two domains (Czerny and Busslinger 1995
). An efficient strategy to overcome this difficulty is presented later and used to identify candidate amino acid changes responsible for the differences in binding properties between different groups of paired domains. The candidate changes are then tested, individually or in combination, by in vitro binding and in vivo functional assays.
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Material and Methods |
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D, , and
(which is inferred as the expected number of changes in the tree, excluding the change under study) can be computed by Gu and Zhang's method (1997)
. The relative rates are then measured by the relative rate score (S) defined by
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cDNA Constructs and Site-Directed Mutagenesis
The cDNA sequences of the ancestral paired domain of supergroup I (ANI), the ancestral paired domain of supergroup II (ANII), and the Pax-6 ancestral paired domain (AN6) were constructed by site-directed mutagenesis. A mouse Pax-2 paired box cDNA (gift from Dr. P. Gruss, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany) was used as the template for reconstructing ANI and ANII sequences (see fig. 1B
for amino acid sequence), and human Pax-6 paired box cDNA (gift from Dr. G. Saunders, University of Texas M. D. Anderson Cancer Center, Houston, Tex.) was used as the template for reconstructing the AN6 sequence (see fig. 1B
). Mutations were introduced into the template by sequential PCR steps. Pairs of complementary oligonucleotides containing the desired point mutations were used with primers flanking the paired box region to amplify overlapping fragments covering the whole paired box region; these fragments were mixed in the same tube and amplified by the flanking primers. The oligonucleotide sequences used are available upon request. The reconstructed paired boxes were cloned into the EcoRI-XhoI sites of the polylinker region of pCITE 4b (+) vector (Novagen, Madison, Wis.). Their sequences were verified and used as templates for in vitro transcription-translation in the TNT T7coupled reticulocyte lysate system (Promega, Madison, Wis.) according to the manufacturer's instructions.
Different variants of the full-length eyeless cDNA for testing in flies were generated by site-directed mutagenesis following the similar steps outlined earlier. Once the full-length cDNAs were constructed, they were cloned in pUAST (Brand and Perrimon 1993
) for introduction into flies. cDNA DP6M3 encodes the full-length Eyeless protein with a single substitution at position 47 of the paired domain from the Pax-6 specific asparagine (N) to the histidine (H) occurring in Pax-2, 5, 8. cDNA M2 encodes the full-length Eyeless protein with the complete Eyeless paired domain substituted with the paired domain of Pax-2. M2M3 corresponds to M2 with a single substitution at position 47 of the paired domain from the histidine (H) occurring in Pax-2, 5, 8 to the Pax-6specific asparagine (N).
Electrophoretic Gel Mobility Shift Assay (EMSA)
The seven test-binding sequences were selected from previously tested sequences: 5S2A (originally named 5s2A) and PRS5 by Czerny, Schaffner, and Busslinger (1993)
; H2A2.2, H2B2.1, and H2B2.2 by Barberis et al. (1989)
; and CD19-1 and CD19-2 by Kozmik et al. (1992)
. The complementary oligonucleotides containing a Pax-binding site were annealed in 2 x SSC-Tris solution. One-hundred-and-eighty femtomoles of annealed probes were labeled with 32P by a Klenow fragment and eluted into 120 µl TE through a G-50 column (Pharmacia, Piscataway, NJ). Two microliters of the labeled probes was used in one binding reaction with paired domain peptides. The amount of peptide in 2 µl of translation mixture was measured with the S-tag rapid kit (Novagene, Madison, Wis.), so that approximately equal amounts of peptide were used in each binding reaction (around 0.30.5 pmol). Peptide was mixed with probe in 6.5% glycerol, 0.5% NP40, 0.5 mg/ml BSA, 0.2 mM DTT, 0.7 mM EDTA, 90 mM KCL, 15 mM Tris-HCl (pH7.5), 1 µg poly[dI-C] in a total volume of 20µl at 25°C for 30 min. The DNA-protein complexes were then resolved in 8% native Long Ranger sequence gels (FMC, Rockland, Me.) containing 0.25% TBE.
In order to measure the difference in DNA-binding affinities between mutant ANI-NVS and ANII-DIN, EMSA was carried out with fixed amounts of protein and radioactive-labeled probe 5S2A with increasing amounts of cold 5S2A (0x, 50x, 100x, 200x, 400x, 1000x, 2000x concentration of the 3 fmollabeled probe). The protein amounts were 0.25 pmol for ANI-NVS and 0.26 pmol for ANII-DIN as measured with the S-tag rapid kit. The shift and free probes were quantitated with a phosphorimager (Molecular Dynamics, Sunnyvale, Calif.). The amount of bound probe was calculated from the distribution of shift versus free probe, and a Scatchard plot was drawn from the average of three repeat experiments. Plotting the ratio of bound probe versus unbound probe against the bound probe gave a linear function. The Y-intercept of the regression line was proportional to P0Kr' and its slope was -Kr', which equals the binding affinity preference divided by the amount of poly[dI-dC]. The molecular concentration of the protein was P0, Kr' was proportional to the affinity of the protein to the probe, and P0 could be estimated from the X-intercept (Calzone et al. 1988
).
Drosophila Transgenics and Genetics
dppblink-GAL4 (Staehling-Hampton et al. 1994
) was used as the driver line to induce ectopic eyes on antennae, legs, and wings. UE10 and UE11 are homozygous viable insertions of UAS-eyeless on chromosomes 3 and 2, respectively (Halder, Callaerts, and Gehring 1995
). Drosophila transformants for UAS-DP6M3, UAS-M2, and UAS-M2M3 were generated using P-elementmediated transformation, essentially as described by Rubin and Spradling (1982)
. The recipient strain for all constructs was y ac w. We obtained several independent transformants for each of the constructs, namely M2.3, M2.9, M2.26, M2M3.31, M2M3.33, DPM3.6, DP6M3.15, and DP6M3.17. Different insertions for a given transgene can result in variations in protein levels when expressed under the control of a particular GAL4 driver line. We corrected for this possible variation in several ways. First, all UAS-transgenes were overexpressed using a single GAL4 driver line, dppblink-GAL4. Second, for the in vivo experiments, we made use of all available independent insertions for individual transgenes to correct for possible inter-strain variability. Third, we evaluated the protein expression levels reached during overexpression of the different transgenic constructs with dppblink-GAL4 by Western blotting combined with densitometry (using NIH Image Software). In brief, by means of four independent Western blots, transgenic protein levels were compared in samples containing protein extracts of wing and leg discs (which expressed the transgenes) of three larvae per sample. Although variation did exist between independent samples of the same transgene, and between samples of different transgenes, the average protein levels were not significantly different, thereby corroborating that the use of multiple different transformant lines per transgene eliminates any existing variation. Lastly, we used large sample sizes to estimate ectopic eye sizes. In conclusion, the differences observed in the experiments between different transgenes were because of the molecular differences (i.e., the site-directed mutations) between them, and not because of positional effects.
Determination of Drosophila Red Eye Pigment Concentration
Determination was carried out as described by Evans and Howells (1978)
. In brief, thoraces from five flies with ectopic eyes on wings and legs were sonicated in 400 µl of a 1:1 mixture of 0.1% NH4OH and chloroform. The samples were then centrifuged for 5 min at 14,000 rpm to remove debris. Two-hundred microliters of the supernatant of each sample was split into two wells of a 96-well plate, and spectrophotometric readings were taken at 485 nm. For each cross, multiple samples of five flies were taken to determine red eye pigment concentrations (see fig. 6C
for details on sample number).
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Results |
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Inference of Candidate Critical Amino Acids
As there are seven differences between ANI and ANII (fig. 1B
) and 19 amino acid differences between AN6 and ANI, it would have been tedious to test all differences for their effects by mutagenesis and binding assays. The second step of our strategy was to infer good candidate sites as follows. We first computed the relative rates of amino acid substitution at the sites that are different between the two groups under study (table 1
). A site that shows a high rate of evolution would not be a good candidate because it would imply either frequent change in binding properties or little functional contribution to binding. We therefore selected only those sites that show very low relative evolutionary rates as candidate sites. For the differences between ANI and ANII, we selected three sites with the lowest rates: 121, 22, and 20 (table 1
); 74 has the same rate as site 20, but it is not conserved within each of the two supergroups and was therefore not selected for testing. In the comparison between AN6 and ANI, sites 44, 47, and 66 have the lowest relative rates (table 1
) and were selected as candidate sites for the binding specificity differences between AN6 and ANI.
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Two Amino Acid Changes are Largely Responsible for the DNA-Binding Property Differences Between ANI and ANII
Figure 3A
shows the effects of single mutations at site 20, 22, or 121 on the binding patterns of ANI and ANII. No single mutation at these three sites changed the binding properties of ANI significantly, as shown in the binding patterns of ANI-NVN (D20N), ANI-DVS (N121S), and ANI-DIN (V22I). However, changes at positions 20 (N20D, ANII-DIS) or 121 (S121N, ANII-NIN) in ANII greatly increased the binding strength of ANII to the test sequences: the binding properties of these ANII variants resembled those of ANI. In contrast, I22V in ANII (ANII-NVS) failed to significantly increase the binding of ANII to any of the test sequences.
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In Vitro and In Vivo Analysis of the Evolutionary Divergence Within Supergroup I: A Single Critical Amino Acid Change Between ANI and AN6
Figure 5
shows the effects of changes (individually or in combination) in ANI from R to Q, H to N, and G to R at positions 44, 47, and 66 (i.e., R44Q, H47N, and G66R), respectively, and the reciprocal alterations in AN6 (i.e., Q44R, N47H, and R66G). The binding pattern of ANI-RNG was very similar to that of AN6, except it also showed weak HPD6-like binding to H2B2.1, whereas the broad binding specificity of AN6-QHR was very similar to that of ANI. This finding suggests that an amino acid change at site 47 (H47N in ANI or N47H in AN6) alone can almost completely swap the binding patterns between ANI and AN6. In comparison, site 44 is less important because the change R44Q (i.e., ANI-QHG) had only a slight quantitative effect on the binding properties of ANI and the reciprocal change Q44R (i.e., AN6-RNR) caused weak binding to H2B2.1 and has only a slight quantitative effect on the binding to CD19.1 and H2B2.2. In combination, R44Q and H47N in ANI (ANI-QNG) showed the same binding properties as AN6, whereas Q44R and N47H in AN6 (AN6-RHR) only had a modest increase in affinity relative to the change of site 47 alone (AN6-QHR). Adding a change at site 66 had a modest quantitative effect on the binding properties of ANI and AN6 (fig. 5
).
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Discussion |
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Because some Pax proteins also contain a homeodomain and because the paired domain and the homeodomain may interact with each other in DNA-binding (Underhill, Vogan, and Gros 1995
; Fortin, Underhill, and Gros 1998
), our study represents a simplified approach to simulate the process of the evolution of Pax DNA-binding properties. Nevertheless, our study has provided novel insight into the evolution of paired domains. The ancestors of supergroups I and II have very different binding properties to the panel of test sequences used in this study, and two amino acid substitutions have dominant effects on swapping their binding properties. Because there is no reliable root to the phylogenetic tree of Pax genes, we cannot predict the sequence of the common ancestor of all Pax genes and its binding properties to complete the whole picture of early Pax evolution. However, it is intriguing to speculate that gene duplication of a common ancestor gave rise to the two ancestors of supergroups I and II, and these two ancestor genes mutated at positions 20 and 121 and acquired different DNA-binding properties to initiate the differentiation of the two supergroups. It is possible that the DNA-binding property of the common ancestor resembles the ANI, and the accumulated mutations in sites 20, 121 (and others) might have gradually changed the protein structure to acquire new DNA-binding properties. As measured by the induction of ectopic eyes, our in vivo data clearly demonstrate that even a complete replacement of the paired domain of Eyeless by the one from Pax-2 does not abolish biological activity. This clearly suggests that in vivo, there is significant flexibility with regard to target-binding site recognition and utilization. At the same time it suggests a possible way through which gradual changes can accumulate without necessarily being detrimental to the organism. In conjunction with a gene duplication, one can envisage that initially the genes largely regulate the same targets but that over time the target gene populations will start to diverge. Lastly, our data indicate that the common ancestor of supergroup I had DNA-binding properties very similar to modern members of Pax-2, 5, 8 such as Pax-2. Also, the inferred ancestor paired domain sequence is very similar to the modern Pax-5 paired domain. Thus, it appears that the Pax-2, 5, 8 group has an ancient origin, and the original function could still be well preserved in some modern Pax-2, 5, 8 group members.
Evolution of the Paired Domain Within Supergroup I: Residue 47 is a Key Factor in the Divergence of the Pax-2, 5, 8 and Pax-6 Groups
Our novel approach using evolutionary analysis, in vitro binding assays, and in vivo ectopic eye induction identified residue 47 of the paired domain as the most critical for the sequence recognition difference between ANI and AN6. Previous structural and in vitro binding studies also identified an essential role for residue 47 (Czerny and Busslinger 1995
; Xu et al. 1995
, 1999
). The consistency of our results with previous reports, as well as the novel residues important for the divergence of the two supergroups (ANI and ANII) identified in the current study, are strong arguments for the validity and potentially wide usefulness of our multidisciplinary approach. Residue 47 is the first in the recognition helix (helix three). In Pax-6 (human), residue 47 is an asparagine (N47), whereas a histidine is present at position 47 in Drosophila Paired (Prd). These residues were shown to interact with DNA in a significantly different manner. H47 in Paired forms hydrogen bonds with a guanine at position 4 of the DNA consensus oligo used for crystallization (Xu et al. 1995
). In contrast, N47 in Pax-6 recognizes an AT basepair by means of a van der Waals contact with a thymine at position 4 and a water-mediated contact with the phosphate of thymine at position 2 (Xu et al. 1999
). The different interactions with DNA form the structural basis for the sequence-specificity observed in the current study, as well as in two other studies. Previously, Jun and Desplan (1996)
demonstrated that reciprocal mutations at residue 47 in the Pax-6 and Prd paired domain were able to change the preferred binding specificity of two consensus binding sequences, PrdL and Pax-6L. Czerny and Busslinger (1995)
had previously shown that a combination of changes at sites 42, 44, and 47 can completely swap the binding patterns of Pax-5 and Pax-6. In the current study, we demonstrated that residue 47 is the most critical difference between Pax-6 and Pax-2, 5, 8 within supergroup ANI. However, our evolutionary analysis revealed that site 42 is not a good candidate site because the rate of evolution at this site is relatively high. In fact, although ANI-RNG, ANI-QNG, and ANI-QNR have Q, whereas AN6 has L at position 42 (fig. 1B
), they show basically the same binding properties (fig. 2
).
As position 47 is mainly responsible for the DNA-binding property divergence of the Pax-6 lineage from the common ancestor of supergroup I, it is interesting to note that Pax-4 also has a N at site 47, further supporting the clustering of Pax-4 with Pax-6 (Balczarek, Lai, and Kumar 1997
). We propose that the substitution H47N occurred early in the duplicated gene of the ancestor of supergroup I and gave rise to the common ancestor of the Pax-6-Pax-4 group. In a later stage, gene duplication and more amino acid substitutions occurred, giving rise to the Pax-6 and Pax-4 lineages. In the formation of the Pax-6 lineage, the substitution R44Q might play a role in further defining the functional specificity of the Pax-6 gene. However, the substitution H47N is the most crucial step in the formation of the Pax-6 lineage. As for the Pax-4 lineage, only position 20 is different between Pax-4 and Pax-6 for all the critical amino acid residues we examined. The fact that most amino acid changes found in Pax-4 are unique implies that the unique property of the Pax-4 paired domain was derived from the common ancestor of Pax-6 and Pax-4. Further efforts to identify critical amino acid substitutions that led to the Pax-4 lineage will complement our knowledge of the functional evolution of this important Pax group.
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Acknowledgements |
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Footnotes |
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Keywords: paired domains
binding assays
in vivo tests
DNA-binding properties
functional evolution
Address for correspondence and reprints: Wen-Hsiung Li, Department of Ecology and Evolution, University of Chicago, 1101 East 57th Street, Chicago, Illinois 60637. E-mail: whli{at}uchicago.edu
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References |
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