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Spatial and temporal expression of Zimp7 and Zimp10 PIAS-like proteins in the developing mouse embryo
Gene Expression Patterns 8 (2008) 206–213 www.elsevier.com/locate/gep
Spatial and temporal expression of Zimp7 and Zimp10 PIAS-like proteins in the developing mouse embryo Hector Rodriguez-Magada´n, Enrique Merino, Denhı´ Schnabel, Laura Ramı´rez, Hilda Lomelı´ * Departamento de Gene´tica del Desarrollo y Fisiologı´a Molecular, Instituto de Biotecnologı´a, Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Morelos 62210, Me´xico Received 10 May 2007; received in revised form 13 October 2007; accepted 15 October 2007 Available online 24 October 2007
Abstract ZIMP7 and ZIMP10 are two novel human PIAS-like proteins that share a similarity beyond the SP-RING Zn-finger domain that characterizes the PIAS family. This extended similarity is conserved in proteins of several other species and define an independent subfamily. ZIMP10 has been shown to increase the sumoylation of the androgen receptor (AR) leading to a stimulation of AR-mediated transcription. The Drosophila tonalli (tna) is the ortholog gene of ZIMP7 and ZIMP10 and presents genetic interactions with the SWI–SNF complex. Mutations in the tna gene produce flies with homeotic phenotypes. In this study, we determined the spatial–temporal expression pattern of Zimp7 and Zimp10 in mouse embryos from embryonic day 7.5 (E7.5), to mid-gestation. We found that these two genes are extensively expressed during these embryonic days and present partially overlapping patterns with a predomination of the transcripts in the neural tissues at early stages and a drop of expression at E12.5. Unlike other PIAS proteins, the tonalli-related Zimp genes might be essential for development. Comparison of conserved motifs in Zimp7 and Zimp10 protein sequences identified characteristic family domains that might be related to their specific biological roles, besides their common role previously identified in the sumoylation pathway. 2007 Elsevier B.V. All rights reserved. Keywords: Zimp proteins; Sumoylation; X-SPRING; Mouse embryos; Tonalli; Phylogenetic analysis; SP-RING; Zn-finger; PIAS-like proteins
1. Results and discussion ZMIZ2 (ZIMP7) and ZMIZ1 (ZIMP10) are PIAS-like proteins that have been described as androgen receptor (AR) co-regulators (Beliakoff and Sun, 2006). Members of the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] family are characterized by the presence of a zinc finger domain termed SP-RING, which has been associated with the sumoylation process. The PIAS proteins were first identified as negative regulators of the JAK-STAT pathway and are evolutionarily conserved from flies to humans. Now, it is established that they participate in a wide variety of signaling path*
Corresponding author. Tel.: +52 73 29 1663; fax: +52 73 17 2388. E-mail address: [email protected] (H. Lomelı´).
1567-133X/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2007.10.005
ways including P53, Wnt and steroid hormone receptor signaling (reviewed in Schmidt and Muller, 2003). In Drosophila, TnaA protein encoded by the tonalli (tna) gene shows important similarity with ZIMP7 and ZIMP10 in a region that extends beyond the SP-RING, suggesting that these Zimp proteins could be orthologs of TnaA. The common domain shared by these three proteins was called X-SPRING (eXtended SP-RING) domain and defines a subfamily of the PIAS group (Gutierrez et al., 2003). The tna gene had been previously identified by its genetic interactions with the SWI/SNF chromatin-remodeling complex (Gutierrez et al., 2003). Recent studies have also demonstrated that ZIMP7 physically interacts with essential components of the SWI/SNF complex, including the catalytic subunit Brg1 (Huang et al., 2005). On the
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Fig. 1. (A) Phylogenetic tree of Zimp proteins. The common names of some organisms are included in parenthesis. The names of the organism with green lettering represent Zimp10 proteins whilst those in blue lettering represent Zimp7. Zimp proteins of Drosophila, Apis and Tribolium represent a third group of Zimp proteins, which are colored in red. (B) Sequence motifs in Zimp proteins. Contiguous over-represented motifs in Zimp proteins were clustered and represented by distinctive colored boxes. The X-SPRING domain including the SP-RING of the PIAS family is boxed in yellow whilst the proline-rich domains are boxed in blue. Color code for Zimp7 (blue), Zimp10 (green) or Drosophila tonalli (tna) group (red), are the same as in (A). The zig-zag lines indicates that a conserved block is interrupted by the insertion of sequence, Pan troglodytes and Gallus gallus Zimp proteins are partially shown. The fulllength representation of these proteins is shown in (C).
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Fig. 2. Logo representation of conserved motifs common to all Zimp proteins. Aligned blocks of contiguous sequences present in Zimp proteins were used as input to the LOGO web server at http://weblogo.berkeley.edu/ (Crooks et al., 2004) to obtain the representation of the relative frequency of each amino acid in the motifs. Note that many positions in these blocks are highly conserved, thus they have the maximum possible frequency value of 100%. Color code representation for Proline-rich region is boxed in blue whilst the X-SPRING domain is boxed in red. The consensus bases of the SP-RING are marked by asterisks. Polar amino acids (G, S, T, Y, C, Q, N) are shown in blue; basic amino acids (K, R, H) are shown in green; acidic residues (D, E) are shown in purple whilst hydrophobic amino acids (A, V, L, I, P, W, F, M) are shown in black. Proline residues are marked in red.
other hand, Sharma et al., 2003 showed that both ZIMP7 and ZIMP10 colocalise with SUMO in the nucleus of human prostate epithelial cells and form a replication complex at replication foci (Sharma et al., 2003). In addition, ZIMP10 was also shown to enhance the sumoylation of the AR and augment its transcriptional activity in several cell lines. These data suggest that ZIMP proteins could be regulating the transcription of specific target genes by altering their chromatin structure through the sumoylation of specific subunits of the SWI/SNF complex. In Drosophila, mutations in the tna gene affect the expression of homeotic genes like Antennapedia, Ultrabithorax and Sex combs reduced (Gutierrez et al., 2003). Also, it was recently reported that ZIMP10 enhanced the transcriptional activity of Smad3 suggesting a connection between ZIMP10 and the TGFb/Smad signaling pathway (Li et al., 2006). These observations together with the involvement of ZIMP7 and ZIMP10 in the transcriptional
regulation of nuclear receptors predict that the mammalian Zimp7 and Zimp10 proteins will show important roles during embryonic development. In the mouse genome Zimp7 and Zimp10 are present and share 87% and 97% of similarity with their corresponding human counterparts. In this study, we analyzed Zimp7 and Zimp10 mRNA expression patterns in mice embryos. We found that the expression patterns of these genes are partially overlapping: at early stages, the transcripts for both of these proteins were strongly enriched in the neural tissue, by mid-gestation the sharpest expression was found with a broad distribution throughout the embryo of both transcripts; after this stage the expression of Zimp7 and Zimp10 mRNAs started to decline and nearly turned off by E12.5 where both transcripts were present in the limbs, somites and few brain regions. This pattern is consistent with a participation of the mouse Zimp proteins during embryonic development.
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1.1. Phylogenetic and structural relationships among X-SPRING subfamily genes of different species In order to have an overall view of the primary structure of the Zimp proteins, a set of 25 non-redundant sequences were aligned using the MUSCLE program (Edgar, 2004). Three main kind of regions in the aligned sequences were identified: (a) Highly conserved regions present in all the set of Zimp7, Zimp10 or the Drosophila melanogaster TnaA protein homologs; (b) Highly conserved regions shared only by Zimp7 or Zimp10 homologs; (c) Not conserved sequence regions. To identify more precisely over-represented motifs in Zimp proteins, we used the public domain motif discovery tool MEME (Bailey and Elkan, 1994). Although many of the conserved blocks identified in our analysis were found to be present in all Zimp proteins, distinctive blocks predominantly found in Zimp7 or Zimp10 were also found. The presence (value 1) or the absence (value 0) of these motifs was utilized in the CLIQUE program (Felsenstein, 1995) to construct a phylogenetic tree (Fig. 1A). Interestingly, this simplified representation of the information carried by Zimp sequences (only 30 binary data per sequence) was sufficient to obtain a congruent tree of this family
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of proteins with Zimp7 and Zimp10 orthologs grouped at distant branches and the Drosophila tonalli (tna) placed in between. In order to simplify the graphical representation of these 30 conserved motifs in Zimp proteins, contiguous blocks were clustered into larger units. Different clusters sharing sequence similarity were identified in this process (Fig 1B and C). The most statistically significant conserved cluster of our analysis included the previously identified X-SPRING domain characteristic to a subgroup of the PIAS protein family (Gutierrez et al., 2003). This region includes the SP-RING zinc finger domain required for PIAS-mediated sumoylation process (Kotaja et al., 2002). The flanking regions of the X-SPRING domain are also highly conserved in all Zimp proteins and together with this domain, form a conserved block greater than 300 amino acids with more than 90% identity (Figs. 1B and 2A). Two more conserved regions, localized at their amino and carboxy-terminus, were found to be shared by all Zimp proteins (Fig. 2B and C). In addition to these conserved regions, the compilation of the Zimp proteins identified two important proline-rich regions. The first proline-rich region is located at positions 334– 555 in ZIMP10 and at 201–403 in ZIMP7. The car-
Fig. 3. Expression of Zimp7 at embryonic stages E7.5 to E9.5. (A–C) Whole mount in situ hybridization of E7.5 (A) E8.5 (B) and (C) embryos probed for Zimp7. (D) Frontal histological section of an embryo at the level indicated in C. (E) E9.0 Zimp7 probed embryo. (F) Transverse histological section of embryo in (E) at the indicated level. (G) Whole mount in situ hybridization of an E9.5 embryo. Al, allantois; ACh, atrial chamber; BA, branchial arches; Ch, chorion; Fb, forebrain; FG, foregut; FL, forelimb; H, heart; Hb, hindbrain; HG, hindgut; HL, hindlimb; Mb, midbrain; N, notochord; Ne, neuroectoderm; NT, neural tissue; OtP, otic pit; PS, primitive streak; S, somites; VCh, ventricular chamber.
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insertion sequences are located disrupting different conserved blocks. Despite these insertions at the amino terminus of P. troglodytes and G. gallus or at different position along the C. familiaris protein, the remaining parts of these proteins are extremely conserved. 1.2. Expression of Zimp7 and Zimp10 mRNAs in gastrulating to mid-gestation mouse embryos
Fig. 4. Expression of Zimp7 at embryonic stages E10.5 and E11.5. (A) Whole mount in situ hybridization of a E10.5 embryo probed for Zimp7. (B) Sagittal histological section of embryo in (A) at a midline level. (C) E9.0 sense probed embryo. (D and E) Whole mount in situ hybridization of E11.5 embryos. BA, branchial arches; Fb, forebrain; FL, forelimb; G, developing gut; H, heart; Hb, hindbrain; HL, hindlimb; LB, lung bud; Mb, midbrain; ML, mesencephalic lobule; NT, neural tissue; OV, optic vesicle; OtV, otic vesicle; SCh, spinal chord; St, stomach; Tel, telencephalon; TV, telencephalic vesicle.
boxy-terminal proline-rich domain corresponds to position 867–1002 in ZIMP10 and 725–906 in ZIMP7. The carboxy-terminal proline-rich region in ZIMP10 has been found to possess a significant intrinsic transcriptional activity that can be inhibited by its interaction with the amino-terminal domain of the protein (Sharma et al., 2003). The modulation of the transcriptional activity of the rat androgen receptor has also been found to be dependent on the interaction of its amino- and carboxyl-terminal regions (Ikonen et al., 1997). On the other hand, the finding that a proline rich domain present in the SUMO E2 conjugase Ubc9 has a critical role in the modulation of the transforming versus growth-promoting properties of the HMGA1b transcription factor suggests that the Zimp proline rich regions might be important for biological activity (Li et al., 2007). The architecture of Zimp10 proteins from Pan troglodytes and Gallus gallus presents special features. These proteins double the size of canonical Zimp10 sequences due to a very large insertion at their amino-terminal regions (Fig. 1C). The extra-region of these proteins might be the result of the rearrangement of their corresponding genes. A different example of non-canonical Zimp protein corresponds to Canis familiaris Zimp7 (Fig. 1B). In this case,
Expression of Zimp7 and Zimp10 genes in mouse embryos was analyzed by whole mount in situ hybridization using cRNA probes. At embryonic day 7.5 (E7.5), Zimp7 transcripts were mainly detected in the primitive streak and at the anterior region of the embryonic ectoderm (presumptive neuroectoderm) (Fig. 3A). A weaker signal was observed in the epiblast and the embryonic mesoderm. Transcripts were not detected in the extraembryonic region except for some signal in the chorion. At E8.5 Zimp7 transcripts were observed in the neural tissue in the hindbrain and midbrain and the anterior mesenchyme extending up to the second anterior somite (Fig. 3B and C). The forebrain region did not show the transcript at this stage (Fig. 3C). Zimp7 transcripts were also detected in the branchial arches, foregut and notochord. Frontal sections confirmed the pattern of expression in the neural tissue and indicated expression in the gut endoderm (Fig. 3D). At E9.0, there were high levels of Zimp7 expression in the anterior region of the embryo, this expression however, was markedly down regulated in the caudal region. (Fig. 3E and F). Transverse sections revealed ubiquitous expression of Zimp7 from top of the head up to the heart level (Fig. 3F). Sites of expression included the developing brain, the head and anterior mesenchyme, the branchial arches and the otic pits. In contrast no transcripts were detected in the ventricular chamber of the heart or in the somites. Zimp7 gene expression continued to extend from anterior to posterior and by E9.5 transcripts were found present in the mesenchyme extending up to the forelimb bud level (Fig. 3G). Both forelimbs and hindlimbs exhibited Zimp7 expression at this stage. At E10.5, whole mount embryos presented a wide expression of the Zimp7 gene (Fig. 4A). The most prominent expression sites were the head and trunk mesenchyme, ventral forebrain regions, maxillar and mandibular components of the branchial arches and the otic and optic vesicles. Sagittal sections of embryos at this stage confirmed these sites of expression and showed additional expression in the developing intestine, stomach and lung bud (Fig. 4B). At 11.5, Zimp7 was sharply down regulated in many regions of the embryo with the exception of the neural tube, brain regions and the limbs (Fig. 4D and E). The neurological expression of Zimp7 displayed sharp boundaries around the telencephalic and mesencephalic vesicles and a very clear signal along the spinal chord (Fig. 4E). A very similar expression pattern is observed at E12.5 (data not shown).
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Fig. 5. Expression of Zimp10 at embryonic stages E7.5 to E9.5. (A and B) Whole mount in situ hybridization of E7.5 (A), E8.5 (B) embryos probed for Zimp10. (C) Transverse histological section of a Zimp10 probed E8.5 embryo. (D) E9.5 sense probed embryo. (E) Cartoon of an E8.5 embryo indicating levels of sections in C and F. (F) Frontal section of a Zimp10 probed E8.5 embryo. (G) Whole mount in situ hybridization of an E9.5 embryo probed for Zimp10. (H) Parasagittal section of embryo in F at a midline level. Al, allantois; BA, branchial arches; BAr, branchial arch artery; CNe, caudal neuroectoderm; DA, dorsal aorta; Fb, forebrain; FG, foregut; Hb, hindbrian; Mb, midbrain; N, notochord; Ne, neuroectoderm; OV, optic vesicle; OtV, otic vesicle; PM, paraxial mesoderm; PS, primitive streak; S, somites; UV, umbilical vein; VE, visceral endoderm.
Similar embryo stages were analyzed with the Zimp10 riboprobe. At E7.5, Zimp10 transcripts were generally observed in the epiblast, but much more abundantly in the anterior portion, at the presumptive neural ectoderm (Fig. 5A). Expression was also detected in the embryonic mesoderm and allantois. At E8.5 Zimp10 expression was largely confined to the neuroectoderm, notochord, dorsal aorta and the allantois (Fig. 5B). The expression in these two latter regions indicated the presence of Zimp10 in the developing vascular system. Histological sections at E8.5 confirmed the presence of Zimp10 mRNA along the neuroectoderm and revealed some expression in the paraxial mesoderm and foregut (Fig. 5C and F). At E9.5 Zimp10 showed a broad distribution throughout the embryo (Fig. 5G) with the most remarkable expression in the neural tissue, the optic vesicle, the otic pit, and the branchial arches. However as in the case of Zimp7 no signal was detected in the ventricular chamber of the heart or in the tail bud. Sagittal sections of these embryos demonstrated that Zimp10 expression was very prominent in the brain and epithelium around the head, the umbilical vein, arteries of the branchial arches and the somites (Fig. 5H). In E10.5 whole mount embryos, the regions that exhibited expres-
sion were the telencephalic and optic vesicles, the mandibular components of branchial arches, the otic vesicles and the somites (Fig. 6A). In addition, Zimp10 was expressed in both the forelimb and hindlimb analogous to Zimp7. Sagittal sections of these embryos demonstrated that Zimp10 transcripts are present in all the brain regions (Fig. 6B). On the other hand at this stage the dorsal aorta did not longer show a positive signal (Fig. 6B). In a similar manner as for the Zimp7 gene, a decrease of the signal for Zimp10 was detected by E11.5. At this stage Zimp10 was restricted to the limb buds, brain regions and the tail bud (Fig. 6C). At E12.5, signal could only be significantly detected in the limbs and tail bud (Fig. 6D), although longer developing times revealed expression in regions of proliferation like the nasal process and the primordia of follicle of vibrissae (not shown). At E13.0 isolated brains probed for Zimp10 indicated some expression in neurological tissues (not shown). 1.3. Dynamic expression of Zimp10 mRNAs in the limb buds After the drop of expression of the Zimp transcripts observed between E10.5 and E11.5, the presence of both
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(S2–S6, as classified by Wanek et al., 1989) a posterior– anterior gradient was observed (Fig. 7). At S2 (Fig. 7A) Zimp10 was broadly expressed in the mesenchyme and a discrete gradient of expression was observed in the posterior part of the limb. At S4 (Fig. 7B) this expression gradient was clearly visible; the expression of Zimp10 was restricted to the posterior mesenchyme and absent from the apical ectodermal ridge. This pattern was the same in the ventral and dorsal sides of the limbs. At S6 (Fig. 7C), the pattern was slightly different with the presence of the Zimp10 signal in the posterior mesenchyme in the prospective metatarsal region and an additional expression forming two stripes in the proximal mesenchyme close to the prospective fibula and tibia. At S8 (Fig. 7D), the expression of Zimp10 transcripts became restricted to the proximal interdigital region up to the metatarsal regions and at S10 (Fig. 7E) Zimp10 expression became restricted from the interdigits to the digits at the forming phalangeal joints. At this stage Zimp10 is clearly absent from the interdigital region. At S12 the expression pattern was the same as at S10 (data not shown). Although at a lower level, Zimp7 was also expressed during limb morphogenesis in a similar pattern (data not shown). The expression of Zimp10 in the limb buds is quite significant and is characterized by the presence of the mRNA during the definition of regions within the limb. Fig. 6. Expression of Zimp10 at embryonic stages E10.5–E12.5. (A) Whole mount in situ hybridization of an E10.5 embryos probed for Zimp10. (B) Parasagittal section of embryo in A at the midline level. (C and D) Whole mount in situ hybridization of E11.5 (C) and E12.5 (D) embryos probed for Zimp10. ACh, atrial chamber; BA, branchial arches; FL, forelimb; FP, foor plate; HL, hindlimb; OV, optical vesicle; OtV, otic vesicle; S, somites; Tb, tail bud; TV, telencephalic vesicle; UV, umbilical vein; V, 4th ventricle; VCh, ventricular chamber.
mRNAs in the limb buds became conspicuous. Particularly Zimp10 showed a dynamic and highly interesting pattern. During mouse limb development at stages 2 through 6
1.4. Conclusions In this study, we examine the spatial and temporal expression pattern as well as the genetic structure of the two Zimp family members, Zimp7 and Zimp10. Both Zimp7 and Zimp10 expression are dynamically regulated from E7.5 to mid-gestation in the developing mouse embryo. While there appears to be some overlap of expression in some areas such as the developing neuroepithelium indicating possible functional redundancy, Zimp7 and Zimp10 are uniquely expressed in some tissues. For example, Zimp10 appears to
Fig. 7. Expression of Zimp10 in the limb buds. Ventral views of whole mount in situ hybridization of limb buds at S2 (A) S4 (B) S6 (C) S8 (D) and S10 (E) [as classified by Wanek et al. (1989)] probed for Zimp10. a, anterior; p, posterior. A gradient of expression is observed from the posterior to the anterior end from stages 2 through stage 6, there is no expression in the apical ectodermal ridge. At stage 8, expression is restricted to the posterior region at the metatarsal limit. At stage 10 the expression is mainly observed in the phalangeal boundaries.
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be present in the developing vascular system at early stages with expression in the dorsal aorta and the allantois. Unlike Zimp7, Zimp10 is also expressed in the paraxial mesoderm and somites. At E7.5, we show that Zimp7 possess restricted expression in the primitive streak, while at mid-gestation we observe expression in the spinal chord and brain lobules. Of interest is the dynamic of expression of Zimp7 that extends from anterior to posterior as development proceeds and suggests a particular transcriptional organization. Functional work done for the Zimp proteins indicates that at least Zimp10 has specific roles during transcriptional regulation (Li et al., 2006). Therefore, considering the strong activity of both genes during embryogenesis it is possible that these two genes are important for development. The analysis of Zimp7 and Zimp10 sequences shows that these proteins share common regions of highly conserved sequence in addition to the SP-RING domain that characterizes the PIAS family. Besides these common conserved regions to all Zimp proteins, Zimp7 and Zimp10 proteins present other sequence motifs specific to each of these variants. The phylogenetic analysis of Zimp proteins locates the previously characterized D. tonalli (tna) between the Zimp7 and Zimp10 groups. 2. Experimental procedures 2.1. Computer analyses Using the D. melanogaster TnaA protein sequence and those from its human homologs, ZIMP7 and ZIMP10, all the available Zimp sequences from the GenBank database were identified and retrieved using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/). Partially sequenced or redundant versions of Zimp proteins were eliminated. After this process, the sequences of 23 Zimp proteins were obtained. In order to identify over-represented ‘signatures’ in our set of Zimp protein sequences, we used the public domain motif discovery tool Multiple EM for Motif Elicitation (MEME) (Bailey and Elkan, 1994). Thirty non-overlapping motifs of amino acid sequence carrying no-indels were identified. The presence (value 1) of absence (value 0) of these motifs in the sequences were used as input of the CLIQUE program of J. Felsentein’s PHYLIP 3.57c phylogeny inference package program (Felsenstein, 1995) to obtain the largest cliques of characters and the suggested tree. For simplicity, cluster of contiguous motifs were grouped and represented by continuous boxes (Fig. 1 B). The alignment of Zimp protein sequences was performed using the MUSCLE program (Edgar, 2004). The SP-RING domain was identified using the Pfam web server at http://www.sanger.ac.uk/Software/Pfam/search.shtml.
2.2. Probe preparation The Zimp10 probe was obtained by subcloning into pKS vector a fragment corresponding to bases 1–500 of the coding sequence from the complete cDNA clone. This vector was linearized with HinDIII for the antisense probe or NotI for the sense probe. The Zimp7 probe, corresponded to bases 532–1098 of the coding sequence subcloned into pKS vector and linearized with HinDIII for the antisense probe and NotI for the sense probe. Both antisense riboprobes were in vitro transcribed with digoxigenin RNA labeling mix and T7 RNA polymerase (Roche), and the sense riboprobes were in vitro transcribed with T3 RNA polymerase (Roche).
2.3. Whole-mount RNA in situ hybridization Whole embryos were fixed overnight in 4% paraformaldehyde (PFA) at 4 C and dehydrated in a graded methanol series prior to storage at 20 C. Whole-mount in situ hybridization was performed as described
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previously (Hogan et al., 1994). Briefly, embryos were rehydrated in a graded methanol series and treated with proteinase K (10 lg/ml) (Invitrogen). Embryos were re-fixed with 0.2% glutaraldehyde/ 4% PFA and washed in PBT (0.1% Tween-20/PBS) prior to hybridization overnight at 68 C. Embryos were washed twice in 2· SSC/0.2% SDS at 70 C, followed by two identical washes in 0.2· SSC/0.2% SDS. Embryos were incubated overnight with anti-digoxigenin antibody (Roche) at 4 C and after several washes with TBST color was developed using NBT/BCIP (Roche).
Acknowledgements We thank Dr. Luke Krebs for critical comments on the manuscript. We also thank Claudia Lomelı´ for assistance in the artwork and Marcela Ramirez and Sergio Gonza´les for assistance with mice. This work was supported by DGAPA-UNAM Grants IN213602-3 and CONACyT 49114 and by a sabbatical fellowship to HL by DGAPA-UNAM. References Bailey, T.L., Elkan, C., 1994. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol 2, 28–36. Beliakoff, J., Sun, Z., 2006. Zimp7 and Zimp10, two novel PIAS-like proteins, function as androgen receptor coregulators. Nucl. Recep. Signal 4, e017. Crooks, G.E., Hon, G., Chandonia, J.M., Brenner, S.E., 2004. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190. Edgar, R.C., 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113. Felsenstein, J., 1995. PHYLIP (Phylogeny Inference Package) version 3.57c. Department of Genetics, University of Washington, Seattle. Gutierrez, L., Zurita, M., Kennison, J.A., Vazquez, M., 2003. The Drosophila trithorax group gene tonalli (tna) interacts genetically with the Brahma remodeling complex and encodes an SP-RING finger protein. Development 130, 343–354. Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the Mouse Embryo: a Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, New York. Huang, C.Y., Beliakoff, J., Li, X., Lee, J., Li, X., Sharma, M., et al., 2005. hZimp7, a novel PIAS-like protein, enhances androgen receptormediated transcription and interacts with SWI/SNF-like BAF complexes. Mol. Endocrinol. 19, 2915–2929. Ikonen, T., Palvimo, J.J., Janne, O.A., 1997. Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. J. Biol. Chem. 272, 29821–29828. Kotaja, N., Karvonen, U., Janne, O.A., Palvimo, J.J., 2002. PIAS proteins modulate transcription factors by functioning as SUMO-1 ligases. Mol. Cell. Biol. 22, 5222–5234. Li, X., Thyssen, G., Beliakoff, J., Sun, Z., 2006. The novel PIAS-like protein hZimp10 enhances Smad transcriptional activity. J. Biol. Chem. 281, 23748–23756. Li, Y., Lu, J., Prochownik, E.V., 2007. Dual role for sumo E2 conjugase UBC9 in modulating the transforming and growth-promoting properties of the HMGA1B architectural transcription factor. J. Biol. Chem. 282, 13363–13371. Schmidt, D., Muller, S., 2003. PIAS/SUMO: new partners in transcriptional regulation. Cell Mol. Life Sci. 60, 2561–2574. Sharma, M., Li, X., Wang, Y., Zarnegar, M., Huang, C.Y., Palvimo, J.J., Lim, B., Sun, Z., 2003. hZimp10 is an androgen receptor co-activator and forms a complex with SUMO-1 at replication foci. EMBO J. 17, 6101–6114. Wanek, N., Muneoka, K., Holler-Dinsmore, G., Burton, R., Bryant, S.V., 1989. A staging system for mouse limb development. J. Exp. Zool. 249, 41–49.
Spatial and temporal expression of Zimp7 and Zimp10 PIAS-like proteins in the developing mouse embryo Hector Rodriguez-Magada´n, Enrique Merino, Denhı´ Schnabel, Laura Ramı´rez, Hilda Lomelı´ * Departamento de Gene´tica del Desarrollo y Fisiologı´a Molecular, Instituto de Biotecnologı´a, Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Morelos 62210, Me´xico Received 10 May 2007; received in revised form 13 October 2007; accepted 15 October 2007 Available online 24 October 2007
Abstract ZIMP7 and ZIMP10 are two novel human PIAS-like proteins that share a similarity beyond the SP-RING Zn-finger domain that characterizes the PIAS family. This extended similarity is conserved in proteins of several other species and define an independent subfamily. ZIMP10 has been shown to increase the sumoylation of the androgen receptor (AR) leading to a stimulation of AR-mediated transcription. The Drosophila tonalli (tna) is the ortholog gene of ZIMP7 and ZIMP10 and presents genetic interactions with the SWI–SNF complex. Mutations in the tna gene produce flies with homeotic phenotypes. In this study, we determined the spatial–temporal expression pattern of Zimp7 and Zimp10 in mouse embryos from embryonic day 7.5 (E7.5), to mid-gestation. We found that these two genes are extensively expressed during these embryonic days and present partially overlapping patterns with a predomination of the transcripts in the neural tissues at early stages and a drop of expression at E12.5. Unlike other PIAS proteins, the tonalli-related Zimp genes might be essential for development. Comparison of conserved motifs in Zimp7 and Zimp10 protein sequences identified characteristic family domains that might be related to their specific biological roles, besides their common role previously identified in the sumoylation pathway. 2007 Elsevier B.V. All rights reserved. Keywords: Zimp proteins; Sumoylation; X-SPRING; Mouse embryos; Tonalli; Phylogenetic analysis; SP-RING; Zn-finger; PIAS-like proteins
1. Results and discussion ZMIZ2 (ZIMP7) and ZMIZ1 (ZIMP10) are PIAS-like proteins that have been described as androgen receptor (AR) co-regulators (Beliakoff and Sun, 2006). Members of the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] family are characterized by the presence of a zinc finger domain termed SP-RING, which has been associated with the sumoylation process. The PIAS proteins were first identified as negative regulators of the JAK-STAT pathway and are evolutionarily conserved from flies to humans. Now, it is established that they participate in a wide variety of signaling path*
Corresponding author. Tel.: +52 73 29 1663; fax: +52 73 17 2388. E-mail address: [email protected] (H. Lomelı´).
1567-133X/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2007.10.005
ways including P53, Wnt and steroid hormone receptor signaling (reviewed in Schmidt and Muller, 2003). In Drosophila, TnaA protein encoded by the tonalli (tna) gene shows important similarity with ZIMP7 and ZIMP10 in a region that extends beyond the SP-RING, suggesting that these Zimp proteins could be orthologs of TnaA. The common domain shared by these three proteins was called X-SPRING (eXtended SP-RING) domain and defines a subfamily of the PIAS group (Gutierrez et al., 2003). The tna gene had been previously identified by its genetic interactions with the SWI/SNF chromatin-remodeling complex (Gutierrez et al., 2003). Recent studies have also demonstrated that ZIMP7 physically interacts with essential components of the SWI/SNF complex, including the catalytic subunit Brg1 (Huang et al., 2005). On the
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Fig. 1. (A) Phylogenetic tree of Zimp proteins. The common names of some organisms are included in parenthesis. The names of the organism with green lettering represent Zimp10 proteins whilst those in blue lettering represent Zimp7. Zimp proteins of Drosophila, Apis and Tribolium represent a third group of Zimp proteins, which are colored in red. (B) Sequence motifs in Zimp proteins. Contiguous over-represented motifs in Zimp proteins were clustered and represented by distinctive colored boxes. The X-SPRING domain including the SP-RING of the PIAS family is boxed in yellow whilst the proline-rich domains are boxed in blue. Color code for Zimp7 (blue), Zimp10 (green) or Drosophila tonalli (tna) group (red), are the same as in (A). The zig-zag lines indicates that a conserved block is interrupted by the insertion of sequence, Pan troglodytes and Gallus gallus Zimp proteins are partially shown. The fulllength representation of these proteins is shown in (C).
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Fig. 2. Logo representation of conserved motifs common to all Zimp proteins. Aligned blocks of contiguous sequences present in Zimp proteins were used as input to the LOGO web server at http://weblogo.berkeley.edu/ (Crooks et al., 2004) to obtain the representation of the relative frequency of each amino acid in the motifs. Note that many positions in these blocks are highly conserved, thus they have the maximum possible frequency value of 100%. Color code representation for Proline-rich region is boxed in blue whilst the X-SPRING domain is boxed in red. The consensus bases of the SP-RING are marked by asterisks. Polar amino acids (G, S, T, Y, C, Q, N) are shown in blue; basic amino acids (K, R, H) are shown in green; acidic residues (D, E) are shown in purple whilst hydrophobic amino acids (A, V, L, I, P, W, F, M) are shown in black. Proline residues are marked in red.
other hand, Sharma et al., 2003 showed that both ZIMP7 and ZIMP10 colocalise with SUMO in the nucleus of human prostate epithelial cells and form a replication complex at replication foci (Sharma et al., 2003). In addition, ZIMP10 was also shown to enhance the sumoylation of the AR and augment its transcriptional activity in several cell lines. These data suggest that ZIMP proteins could be regulating the transcription of specific target genes by altering their chromatin structure through the sumoylation of specific subunits of the SWI/SNF complex. In Drosophila, mutations in the tna gene affect the expression of homeotic genes like Antennapedia, Ultrabithorax and Sex combs reduced (Gutierrez et al., 2003). Also, it was recently reported that ZIMP10 enhanced the transcriptional activity of Smad3 suggesting a connection between ZIMP10 and the TGFb/Smad signaling pathway (Li et al., 2006). These observations together with the involvement of ZIMP7 and ZIMP10 in the transcriptional
regulation of nuclear receptors predict that the mammalian Zimp7 and Zimp10 proteins will show important roles during embryonic development. In the mouse genome Zimp7 and Zimp10 are present and share 87% and 97% of similarity with their corresponding human counterparts. In this study, we analyzed Zimp7 and Zimp10 mRNA expression patterns in mice embryos. We found that the expression patterns of these genes are partially overlapping: at early stages, the transcripts for both of these proteins were strongly enriched in the neural tissue, by mid-gestation the sharpest expression was found with a broad distribution throughout the embryo of both transcripts; after this stage the expression of Zimp7 and Zimp10 mRNAs started to decline and nearly turned off by E12.5 where both transcripts were present in the limbs, somites and few brain regions. This pattern is consistent with a participation of the mouse Zimp proteins during embryonic development.
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1.1. Phylogenetic and structural relationships among X-SPRING subfamily genes of different species In order to have an overall view of the primary structure of the Zimp proteins, a set of 25 non-redundant sequences were aligned using the MUSCLE program (Edgar, 2004). Three main kind of regions in the aligned sequences were identified: (a) Highly conserved regions present in all the set of Zimp7, Zimp10 or the Drosophila melanogaster TnaA protein homologs; (b) Highly conserved regions shared only by Zimp7 or Zimp10 homologs; (c) Not conserved sequence regions. To identify more precisely over-represented motifs in Zimp proteins, we used the public domain motif discovery tool MEME (Bailey and Elkan, 1994). Although many of the conserved blocks identified in our analysis were found to be present in all Zimp proteins, distinctive blocks predominantly found in Zimp7 or Zimp10 were also found. The presence (value 1) or the absence (value 0) of these motifs was utilized in the CLIQUE program (Felsenstein, 1995) to construct a phylogenetic tree (Fig. 1A). Interestingly, this simplified representation of the information carried by Zimp sequences (only 30 binary data per sequence) was sufficient to obtain a congruent tree of this family
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of proteins with Zimp7 and Zimp10 orthologs grouped at distant branches and the Drosophila tonalli (tna) placed in between. In order to simplify the graphical representation of these 30 conserved motifs in Zimp proteins, contiguous blocks were clustered into larger units. Different clusters sharing sequence similarity were identified in this process (Fig 1B and C). The most statistically significant conserved cluster of our analysis included the previously identified X-SPRING domain characteristic to a subgroup of the PIAS protein family (Gutierrez et al., 2003). This region includes the SP-RING zinc finger domain required for PIAS-mediated sumoylation process (Kotaja et al., 2002). The flanking regions of the X-SPRING domain are also highly conserved in all Zimp proteins and together with this domain, form a conserved block greater than 300 amino acids with more than 90% identity (Figs. 1B and 2A). Two more conserved regions, localized at their amino and carboxy-terminus, were found to be shared by all Zimp proteins (Fig. 2B and C). In addition to these conserved regions, the compilation of the Zimp proteins identified two important proline-rich regions. The first proline-rich region is located at positions 334– 555 in ZIMP10 and at 201–403 in ZIMP7. The car-
Fig. 3. Expression of Zimp7 at embryonic stages E7.5 to E9.5. (A–C) Whole mount in situ hybridization of E7.5 (A) E8.5 (B) and (C) embryos probed for Zimp7. (D) Frontal histological section of an embryo at the level indicated in C. (E) E9.0 Zimp7 probed embryo. (F) Transverse histological section of embryo in (E) at the indicated level. (G) Whole mount in situ hybridization of an E9.5 embryo. Al, allantois; ACh, atrial chamber; BA, branchial arches; Ch, chorion; Fb, forebrain; FG, foregut; FL, forelimb; H, heart; Hb, hindbrain; HG, hindgut; HL, hindlimb; Mb, midbrain; N, notochord; Ne, neuroectoderm; NT, neural tissue; OtP, otic pit; PS, primitive streak; S, somites; VCh, ventricular chamber.
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insertion sequences are located disrupting different conserved blocks. Despite these insertions at the amino terminus of P. troglodytes and G. gallus or at different position along the C. familiaris protein, the remaining parts of these proteins are extremely conserved. 1.2. Expression of Zimp7 and Zimp10 mRNAs in gastrulating to mid-gestation mouse embryos
Fig. 4. Expression of Zimp7 at embryonic stages E10.5 and E11.5. (A) Whole mount in situ hybridization of a E10.5 embryo probed for Zimp7. (B) Sagittal histological section of embryo in (A) at a midline level. (C) E9.0 sense probed embryo. (D and E) Whole mount in situ hybridization of E11.5 embryos. BA, branchial arches; Fb, forebrain; FL, forelimb; G, developing gut; H, heart; Hb, hindbrain; HL, hindlimb; LB, lung bud; Mb, midbrain; ML, mesencephalic lobule; NT, neural tissue; OV, optic vesicle; OtV, otic vesicle; SCh, spinal chord; St, stomach; Tel, telencephalon; TV, telencephalic vesicle.
boxy-terminal proline-rich domain corresponds to position 867–1002 in ZIMP10 and 725–906 in ZIMP7. The carboxy-terminal proline-rich region in ZIMP10 has been found to possess a significant intrinsic transcriptional activity that can be inhibited by its interaction with the amino-terminal domain of the protein (Sharma et al., 2003). The modulation of the transcriptional activity of the rat androgen receptor has also been found to be dependent on the interaction of its amino- and carboxyl-terminal regions (Ikonen et al., 1997). On the other hand, the finding that a proline rich domain present in the SUMO E2 conjugase Ubc9 has a critical role in the modulation of the transforming versus growth-promoting properties of the HMGA1b transcription factor suggests that the Zimp proline rich regions might be important for biological activity (Li et al., 2007). The architecture of Zimp10 proteins from Pan troglodytes and Gallus gallus presents special features. These proteins double the size of canonical Zimp10 sequences due to a very large insertion at their amino-terminal regions (Fig. 1C). The extra-region of these proteins might be the result of the rearrangement of their corresponding genes. A different example of non-canonical Zimp protein corresponds to Canis familiaris Zimp7 (Fig. 1B). In this case,
Expression of Zimp7 and Zimp10 genes in mouse embryos was analyzed by whole mount in situ hybridization using cRNA probes. At embryonic day 7.5 (E7.5), Zimp7 transcripts were mainly detected in the primitive streak and at the anterior region of the embryonic ectoderm (presumptive neuroectoderm) (Fig. 3A). A weaker signal was observed in the epiblast and the embryonic mesoderm. Transcripts were not detected in the extraembryonic region except for some signal in the chorion. At E8.5 Zimp7 transcripts were observed in the neural tissue in the hindbrain and midbrain and the anterior mesenchyme extending up to the second anterior somite (Fig. 3B and C). The forebrain region did not show the transcript at this stage (Fig. 3C). Zimp7 transcripts were also detected in the branchial arches, foregut and notochord. Frontal sections confirmed the pattern of expression in the neural tissue and indicated expression in the gut endoderm (Fig. 3D). At E9.0, there were high levels of Zimp7 expression in the anterior region of the embryo, this expression however, was markedly down regulated in the caudal region. (Fig. 3E and F). Transverse sections revealed ubiquitous expression of Zimp7 from top of the head up to the heart level (Fig. 3F). Sites of expression included the developing brain, the head and anterior mesenchyme, the branchial arches and the otic pits. In contrast no transcripts were detected in the ventricular chamber of the heart or in the somites. Zimp7 gene expression continued to extend from anterior to posterior and by E9.5 transcripts were found present in the mesenchyme extending up to the forelimb bud level (Fig. 3G). Both forelimbs and hindlimbs exhibited Zimp7 expression at this stage. At E10.5, whole mount embryos presented a wide expression of the Zimp7 gene (Fig. 4A). The most prominent expression sites were the head and trunk mesenchyme, ventral forebrain regions, maxillar and mandibular components of the branchial arches and the otic and optic vesicles. Sagittal sections of embryos at this stage confirmed these sites of expression and showed additional expression in the developing intestine, stomach and lung bud (Fig. 4B). At 11.5, Zimp7 was sharply down regulated in many regions of the embryo with the exception of the neural tube, brain regions and the limbs (Fig. 4D and E). The neurological expression of Zimp7 displayed sharp boundaries around the telencephalic and mesencephalic vesicles and a very clear signal along the spinal chord (Fig. 4E). A very similar expression pattern is observed at E12.5 (data not shown).
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Fig. 5. Expression of Zimp10 at embryonic stages E7.5 to E9.5. (A and B) Whole mount in situ hybridization of E7.5 (A), E8.5 (B) embryos probed for Zimp10. (C) Transverse histological section of a Zimp10 probed E8.5 embryo. (D) E9.5 sense probed embryo. (E) Cartoon of an E8.5 embryo indicating levels of sections in C and F. (F) Frontal section of a Zimp10 probed E8.5 embryo. (G) Whole mount in situ hybridization of an E9.5 embryo probed for Zimp10. (H) Parasagittal section of embryo in F at a midline level. Al, allantois; BA, branchial arches; BAr, branchial arch artery; CNe, caudal neuroectoderm; DA, dorsal aorta; Fb, forebrain; FG, foregut; Hb, hindbrian; Mb, midbrain; N, notochord; Ne, neuroectoderm; OV, optic vesicle; OtV, otic vesicle; PM, paraxial mesoderm; PS, primitive streak; S, somites; UV, umbilical vein; VE, visceral endoderm.
Similar embryo stages were analyzed with the Zimp10 riboprobe. At E7.5, Zimp10 transcripts were generally observed in the epiblast, but much more abundantly in the anterior portion, at the presumptive neural ectoderm (Fig. 5A). Expression was also detected in the embryonic mesoderm and allantois. At E8.5 Zimp10 expression was largely confined to the neuroectoderm, notochord, dorsal aorta and the allantois (Fig. 5B). The expression in these two latter regions indicated the presence of Zimp10 in the developing vascular system. Histological sections at E8.5 confirmed the presence of Zimp10 mRNA along the neuroectoderm and revealed some expression in the paraxial mesoderm and foregut (Fig. 5C and F). At E9.5 Zimp10 showed a broad distribution throughout the embryo (Fig. 5G) with the most remarkable expression in the neural tissue, the optic vesicle, the otic pit, and the branchial arches. However as in the case of Zimp7 no signal was detected in the ventricular chamber of the heart or in the tail bud. Sagittal sections of these embryos demonstrated that Zimp10 expression was very prominent in the brain and epithelium around the head, the umbilical vein, arteries of the branchial arches and the somites (Fig. 5H). In E10.5 whole mount embryos, the regions that exhibited expres-
sion were the telencephalic and optic vesicles, the mandibular components of branchial arches, the otic vesicles and the somites (Fig. 6A). In addition, Zimp10 was expressed in both the forelimb and hindlimb analogous to Zimp7. Sagittal sections of these embryos demonstrated that Zimp10 transcripts are present in all the brain regions (Fig. 6B). On the other hand at this stage the dorsal aorta did not longer show a positive signal (Fig. 6B). In a similar manner as for the Zimp7 gene, a decrease of the signal for Zimp10 was detected by E11.5. At this stage Zimp10 was restricted to the limb buds, brain regions and the tail bud (Fig. 6C). At E12.5, signal could only be significantly detected in the limbs and tail bud (Fig. 6D), although longer developing times revealed expression in regions of proliferation like the nasal process and the primordia of follicle of vibrissae (not shown). At E13.0 isolated brains probed for Zimp10 indicated some expression in neurological tissues (not shown). 1.3. Dynamic expression of Zimp10 mRNAs in the limb buds After the drop of expression of the Zimp transcripts observed between E10.5 and E11.5, the presence of both
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(S2–S6, as classified by Wanek et al., 1989) a posterior– anterior gradient was observed (Fig. 7). At S2 (Fig. 7A) Zimp10 was broadly expressed in the mesenchyme and a discrete gradient of expression was observed in the posterior part of the limb. At S4 (Fig. 7B) this expression gradient was clearly visible; the expression of Zimp10 was restricted to the posterior mesenchyme and absent from the apical ectodermal ridge. This pattern was the same in the ventral and dorsal sides of the limbs. At S6 (Fig. 7C), the pattern was slightly different with the presence of the Zimp10 signal in the posterior mesenchyme in the prospective metatarsal region and an additional expression forming two stripes in the proximal mesenchyme close to the prospective fibula and tibia. At S8 (Fig. 7D), the expression of Zimp10 transcripts became restricted to the proximal interdigital region up to the metatarsal regions and at S10 (Fig. 7E) Zimp10 expression became restricted from the interdigits to the digits at the forming phalangeal joints. At this stage Zimp10 is clearly absent from the interdigital region. At S12 the expression pattern was the same as at S10 (data not shown). Although at a lower level, Zimp7 was also expressed during limb morphogenesis in a similar pattern (data not shown). The expression of Zimp10 in the limb buds is quite significant and is characterized by the presence of the mRNA during the definition of regions within the limb. Fig. 6. Expression of Zimp10 at embryonic stages E10.5–E12.5. (A) Whole mount in situ hybridization of an E10.5 embryos probed for Zimp10. (B) Parasagittal section of embryo in A at the midline level. (C and D) Whole mount in situ hybridization of E11.5 (C) and E12.5 (D) embryos probed for Zimp10. ACh, atrial chamber; BA, branchial arches; FL, forelimb; FP, foor plate; HL, hindlimb; OV, optical vesicle; OtV, otic vesicle; S, somites; Tb, tail bud; TV, telencephalic vesicle; UV, umbilical vein; V, 4th ventricle; VCh, ventricular chamber.
mRNAs in the limb buds became conspicuous. Particularly Zimp10 showed a dynamic and highly interesting pattern. During mouse limb development at stages 2 through 6
1.4. Conclusions In this study, we examine the spatial and temporal expression pattern as well as the genetic structure of the two Zimp family members, Zimp7 and Zimp10. Both Zimp7 and Zimp10 expression are dynamically regulated from E7.5 to mid-gestation in the developing mouse embryo. While there appears to be some overlap of expression in some areas such as the developing neuroepithelium indicating possible functional redundancy, Zimp7 and Zimp10 are uniquely expressed in some tissues. For example, Zimp10 appears to
Fig. 7. Expression of Zimp10 in the limb buds. Ventral views of whole mount in situ hybridization of limb buds at S2 (A) S4 (B) S6 (C) S8 (D) and S10 (E) [as classified by Wanek et al. (1989)] probed for Zimp10. a, anterior; p, posterior. A gradient of expression is observed from the posterior to the anterior end from stages 2 through stage 6, there is no expression in the apical ectodermal ridge. At stage 8, expression is restricted to the posterior region at the metatarsal limit. At stage 10 the expression is mainly observed in the phalangeal boundaries.
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be present in the developing vascular system at early stages with expression in the dorsal aorta and the allantois. Unlike Zimp7, Zimp10 is also expressed in the paraxial mesoderm and somites. At E7.5, we show that Zimp7 possess restricted expression in the primitive streak, while at mid-gestation we observe expression in the spinal chord and brain lobules. Of interest is the dynamic of expression of Zimp7 that extends from anterior to posterior as development proceeds and suggests a particular transcriptional organization. Functional work done for the Zimp proteins indicates that at least Zimp10 has specific roles during transcriptional regulation (Li et al., 2006). Therefore, considering the strong activity of both genes during embryogenesis it is possible that these two genes are important for development. The analysis of Zimp7 and Zimp10 sequences shows that these proteins share common regions of highly conserved sequence in addition to the SP-RING domain that characterizes the PIAS family. Besides these common conserved regions to all Zimp proteins, Zimp7 and Zimp10 proteins present other sequence motifs specific to each of these variants. The phylogenetic analysis of Zimp proteins locates the previously characterized D. tonalli (tna) between the Zimp7 and Zimp10 groups. 2. Experimental procedures 2.1. Computer analyses Using the D. melanogaster TnaA protein sequence and those from its human homologs, ZIMP7 and ZIMP10, all the available Zimp sequences from the GenBank database were identified and retrieved using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/). Partially sequenced or redundant versions of Zimp proteins were eliminated. After this process, the sequences of 23 Zimp proteins were obtained. In order to identify over-represented ‘signatures’ in our set of Zimp protein sequences, we used the public domain motif discovery tool Multiple EM for Motif Elicitation (MEME) (Bailey and Elkan, 1994). Thirty non-overlapping motifs of amino acid sequence carrying no-indels were identified. The presence (value 1) of absence (value 0) of these motifs in the sequences were used as input of the CLIQUE program of J. Felsentein’s PHYLIP 3.57c phylogeny inference package program (Felsenstein, 1995) to obtain the largest cliques of characters and the suggested tree. For simplicity, cluster of contiguous motifs were grouped and represented by continuous boxes (Fig. 1 B). The alignment of Zimp protein sequences was performed using the MUSCLE program (Edgar, 2004). The SP-RING domain was identified using the Pfam web server at http://www.sanger.ac.uk/Software/Pfam/search.shtml.
2.2. Probe preparation The Zimp10 probe was obtained by subcloning into pKS vector a fragment corresponding to bases 1–500 of the coding sequence from the complete cDNA clone. This vector was linearized with HinDIII for the antisense probe or NotI for the sense probe. The Zimp7 probe, corresponded to bases 532–1098 of the coding sequence subcloned into pKS vector and linearized with HinDIII for the antisense probe and NotI for the sense probe. Both antisense riboprobes were in vitro transcribed with digoxigenin RNA labeling mix and T7 RNA polymerase (Roche), and the sense riboprobes were in vitro transcribed with T3 RNA polymerase (Roche).
2.3. Whole-mount RNA in situ hybridization Whole embryos were fixed overnight in 4% paraformaldehyde (PFA) at 4 C and dehydrated in a graded methanol series prior to storage at 20 C. Whole-mount in situ hybridization was performed as described
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previously (Hogan et al., 1994). Briefly, embryos were rehydrated in a graded methanol series and treated with proteinase K (10 lg/ml) (Invitrogen). Embryos were re-fixed with 0.2% glutaraldehyde/ 4% PFA and washed in PBT (0.1% Tween-20/PBS) prior to hybridization overnight at 68 C. Embryos were washed twice in 2· SSC/0.2% SDS at 70 C, followed by two identical washes in 0.2· SSC/0.2% SDS. Embryos were incubated overnight with anti-digoxigenin antibody (Roche) at 4 C and after several washes with TBST color was developed using NBT/BCIP (Roche).
Acknowledgements We thank Dr. Luke Krebs for critical comments on the manuscript. We also thank Claudia Lomelı´ for assistance in the artwork and Marcela Ramirez and Sergio Gonza´les for assistance with mice. This work was supported by DGAPA-UNAM Grants IN213602-3 and CONACyT 49114 and by a sabbatical fellowship to HL by DGAPA-UNAM. References Bailey, T.L., Elkan, C., 1994. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol 2, 28–36. Beliakoff, J., Sun, Z., 2006. Zimp7 and Zimp10, two novel PIAS-like proteins, function as androgen receptor coregulators. Nucl. Recep. Signal 4, e017. Crooks, G.E., Hon, G., Chandonia, J.M., Brenner, S.E., 2004. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190. Edgar, R.C., 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113. Felsenstein, J., 1995. PHYLIP (Phylogeny Inference Package) version 3.57c. Department of Genetics, University of Washington, Seattle. Gutierrez, L., Zurita, M., Kennison, J.A., Vazquez, M., 2003. The Drosophila trithorax group gene tonalli (tna) interacts genetically with the Brahma remodeling complex and encodes an SP-RING finger protein. Development 130, 343–354. Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the Mouse Embryo: a Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, New York. Huang, C.Y., Beliakoff, J., Li, X., Lee, J., Li, X., Sharma, M., et al., 2005. hZimp7, a novel PIAS-like protein, enhances androgen receptormediated transcription and interacts with SWI/SNF-like BAF complexes. Mol. Endocrinol. 19, 2915–2929. Ikonen, T., Palvimo, J.J., Janne, O.A., 1997. Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. J. Biol. Chem. 272, 29821–29828. Kotaja, N., Karvonen, U., Janne, O.A., Palvimo, J.J., 2002. PIAS proteins modulate transcription factors by functioning as SUMO-1 ligases. Mol. Cell. Biol. 22, 5222–5234. Li, X., Thyssen, G., Beliakoff, J., Sun, Z., 2006. The novel PIAS-like protein hZimp10 enhances Smad transcriptional activity. J. Biol. Chem. 281, 23748–23756. Li, Y., Lu, J., Prochownik, E.V., 2007. Dual role for sumo E2 conjugase UBC9 in modulating the transforming and growth-promoting properties of the HMGA1B architectural transcription factor. J. Biol. Chem. 282, 13363–13371. Schmidt, D., Muller, S., 2003. PIAS/SUMO: new partners in transcriptional regulation. Cell Mol. Life Sci. 60, 2561–2574. Sharma, M., Li, X., Wang, Y., Zarnegar, M., Huang, C.Y., Palvimo, J.J., Lim, B., Sun, Z., 2003. hZimp10 is an androgen receptor co-activator and forms a complex with SUMO-1 at replication foci. EMBO J. 17, 6101–6114. Wanek, N., Muneoka, K., Holler-Dinsmore, G., Burton, R., Bryant, S.V., 1989. A staging system for mouse limb development. J. Exp. Zool. 249, 41–49.