Transcriptional repression and developmental functions of the atypical vertebrate GATA protein TRPS1
2001; Springer Nature; Volume: 20; Issue: 7 Linguagem: Inglês
10.1093/emboj/20.7.1715
ISSN1460-2075
Autores Tópico(s)Congenital Ear and Nasal Anomalies
ResumoArticle2 April 2001free access Transcriptional repression and developmental functions of the atypical vertebrate GATA protein TRPS1 Talat H. Malik Talat H. Malik Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Sarah A. Shoichet Sarah A. Shoichet Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Peter Latham Peter Latham Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Todd G. Kroll Todd G. Kroll Department of Pathology, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Luanne L. Peters Luanne L. Peters The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Ramesh A. Shivdasani Corresponding Author Ramesh A. Shivdasani Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Talat H. Malik Talat H. Malik Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Sarah A. Shoichet Sarah A. Shoichet Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Peter Latham Peter Latham Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Todd G. Kroll Todd G. Kroll Department of Pathology, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Luanne L. Peters Luanne L. Peters The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Ramesh A. Shivdasani Corresponding Author Ramesh A. Shivdasani Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Talat H. Malik1,2,3, Sarah A. Shoichet1,2,3, Peter Latham1,2,3, Todd G. Kroll4, Luanne L. Peters5 and Ramesh A. Shivdasani 1,2,3 1Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 2Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA 3Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA 4Department of Pathology, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA 5The Jackson Laboratory, Bar Harbor, ME, USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1715-1725https://doi.org/10.1093/emboj/20.7.1715 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Known vertebrate GATA proteins contain two zinc fingers and are required in development, whereas invertebrates express a class of essential proteins containing one GATA-type zinc finger. We isolated the gene encoding TRPS1, a vertebrate protein with a single GATA-type zinc finger. TRPS1 is highly conserved between Xenopus and mammals, and the human gene is implicated in dominantly inherited tricho-rhino-phalangeal (TRP) syndromes. TRPS1 is a nuclear protein that binds GATA sequences but fails to transactivate a GATA-dependent reporter. Instead, TRPS1 potently and specifically represses transcriptional activation mediated by other GATA factors. Repression does not occur from competition for DNA binding and depends on a C-terminal region related to repressive domains found in Ikaros proteins. During mouse development, TRPS1 expression is prominent in sites showing pathology in TRP syndromes, which are thought to result from TRPS1 haploinsufficiency. We show instead that truncating mutations identified in patients encode dominant inhibitors of wild-type TRPS1 function, suggesting an alternative mechanism for the disease. TRPS1 is the first example of a GATA protein with intrinsic transcriptional repression activity and possibly a negative regulator of GATA-dependent processes in vertebrate development. Introduction Zinc finger transcription factors of the GATA family execute critical, non-redundant functions in vertebrate and invertebrate development. The defining feature of this family is the presence of one or two zinc fingers with the consensus sequence CXNCX17CNXC. One of these motifs binds preferentially to the cognate DNA sequence WGATAR and related sequences found in the control regions of numerous lineage-restricted and developmentally regulated genes (Ko and Engel, 1993; Merika and Orkin, 1993). The six known vertebrate GATA-binding proteins are highly homologous and conserved, contain two GATA-type zinc finger domains, and fall into two subgroups. GATA-1, -2 and -3 are expressed primarily in blood cell lineages and regulate key aspects of hematopoiesis (Pevny et al., 1991; Tsai et al., 1994; Ting et al., 1996; Shivdasani et al., 1997). GATA-4, -5 and -6 are expressed principally in the heart and gut, and their germline absence results in early embryonic defects in heart formation and endoderm development (Laverriere et al., 1994; Kuo et al., 1997; Molkentin et al., 1997; Koutsourakis et al., 1999; Reiter et al., 1999). Phenotypes resulting from GATA factor deficiency in mice, flies and worms underscore the central role of this gene family in cell differentiation and development (Reuter, 1994; Shivdasani and Orkin, 1996; Zhu et al., 1997; Fukushige et al., 1998; Molkentin, 2000). The C-terminal zinc finger of vertebrate GATA proteins binds DNA (Yang and Evans, 1992; Omichinski et al., 1993), whereas the N-terminal finger is implicated both in DNA binding and in mediating protein–protein interactions (Martin and Orkin, 1990; Yang and Evans, 1992; Tsang et al., 1997). In lower eukaryotes proteins of this family typically harbor a single GATA-type zinc finger, as exemplified by Caenorhabditis elegans END-1 and ELT-2 and Drosophila SERPENT/dGATAb, which are required in the earliest stages of endoderm development (Reuter, 1994; Zhu et al., 1997; Fukushige et al., 1998). Nearly complete genome sequences reveal 11 presumptive GATA proteins in C.elegans and four in Drosophila. In each case, the single GATA-type zinc finger is most homologous to the C-terminal finger of vertebrate GATA factors. The N-terminal zinc fingers of vertebrate GATA proteins interact with a variety of nuclear proteins, including the multi-type zinc finger proteins FOG-1 and FOG-2 (Tsang et al., 1997; Holmes et al., 1999; Svensson et al., 1999; Tevosian et al., 1999). These proteins are co-expressed with various GATA factors and the phenotypes of knockout mice reveal essential, GATA-dependent roles in the development of blood and heart cell lineages (Tsang et al., 1998; Svensson et al., 2000b; Tevosian et al., 2000). FOG-1 and FOG-2 demonstrate activity as transcriptional co-activators in some settings and as co-repressors in others (Tsang et al., 1997; Holmes et al., 1999; Tevosian et al., 1999; Deconinck et al., 2000; Svensson et al., 2000a). Similarly, the FOG-related protein U-SHAPED functions in conjunction with the Drosophila GATA factor PANNIER/dGATAa in neural development (Cubadda et al., 1997; Haenlin et al., 1997). Thus, like many other transcription factors, GATA proteins appear to function as DNA-binding components of larger multiprotein complexes. The abundance of GATA sequences in cis-regulatory elements suggests a requirement for added complexity in regulating spatio-temporal expression of lineage-restricted genes. The structural differences between invertebrate and the known vertebrate GATA proteins prompted us to search for atypical vertebrate GATA factors and to study their roles in transcriptional regulation and development. We have assembled full-length cDNAs encoding a vertebrate protein, TRPS1, which contains a single GATA-type zinc finger. Besides this novel feature, TRPS1 differs from all other vertebrate GATA proteins in that it does not function as a transcriptional activator but rather as a potent, sequence-specific transcriptional repressor in vitro and in vivo. TRPS1 is associated with a dominantly inherited human disease of skeletal malformation (Momeni et al., 2000), attesting to its importance in development, and we show that its expression pattern accurately reflects the pathology in affected patients. We also demonstrate that mutations identified in independent kindreds encode truncated proteins that function as dominant antagonists of TRPS1 transcriptional repression activity. Our findings suggest that TRPS1 may act to restrict expression of GATA-regulated genes at selected sites and stages in vertebrate development. Results Isolation of a conserved vertebrate GATA factor representing a new class To isolate members of a potential class of vertebrate proteins with a single GATA-type zinc finger, we performed degenerate RT–PCR on early-stage Xenopus embryos using primers corresponding to the highly conserved C-terminal zinc finger of all GATA proteins. All 24 sequenced PCR products encoded GATA-type zinc fingers, but only one of these encoded the peptide fragment (KNANGGYV) of a novel protein. We used this novel 24 bp PCR product to screen a Xenopus maternal cDNA library and recovered a single partial clone corresponding to a novel GATA protein. Subsequently, we used 5′ rapid amplification of cDNA ends (RACE) and additional library screening to assemble the full-length cDNA. We also compared the Xenopus sequence to identify one mouse expressed sequence tag (EST) clone, used the insert to isolate two partial clones from an embryonic gut cDNA library, and amplified these by 5′ RACE to assemble a full-length homologous murine cDNA. In the course of this isolation, monoallelic mutations in the human homolog of this gene were reported to cause a rare inherited disorder of skeletal malformations, the tricho-rhino-phalangeal (Langer–Giedion) syndrome (TRPS) type I (Momeni et al., 2000). We therefore designate the novel gene products TRPS1. Xenopus (X) and mouse (m) TRPS1 encode proteins of 1272 and 1282 amino acids and show 73 and 93% sequence similarity to the human protein, respectively (Figure 1). TRPS1 includes nine putative zinc finger motifs, only one of which (#7) is of the GATA type. The sequence is 100% conserved in this and in the adjacent basic region, which are required for GATA proteins to bind DNA (Omichinski et al., 1993). The two most C-terminal zinc finger motifs (#8 and #9) and flanking residues constitute a conserved domain found within the Ikaros family of lymphoid transcription factors (Georgopoulos et al., 1997); the remaining sequence lacks homology with known vertebrate or invertebrate proteins. Figure 1.TRPS1 is a highly conserved and atypical vertebrate GATA protein. Deduced amino acid sequences of Xenopus, mouse and human TRPS1. Xenopus and mouse cDNAs were isolated as described; the human sequence is based in part on confirmed EST clones and in part on recently published data (Momeni et al., 2000). Identical amino acids are shaded in black, and conservative substitutions in gray. Positions of the nine putative zinc finger motifs are indicated by arrows under the sequence, the GATA-type zinc finger (residues 886–910 in XTRPS1) is marked by a thick arrow, and the two putative nuclear localization signals are marked by shaded boxes. Download figure Download PowerPoint The form of XTRPS1 expressed early in Xenopus embryos as a maternal transcript lacks amino acids 362–617 (data not shown). These residues are present both in mTRPS1 and in the later-expressed (zygotic) XTRPS1 isoform shown in Figure 1. In the following experiments, XTRPS1 constructs encode the early maternal product; notably, we detect no differences in function between mTRPS1 and this XTRPS1 isoform. TRPS1 is a sequence-specific, DNA-binding nuclear protein TRPS1 includes two conserved nuclear localization signals flanking the GATA-type zinc finger (Figure 1). To determine whether the protein can enter the cell nucleus, we transfected COS cells with plasmids encoding mTRPS1 or the maternally expressed form of XTRPS1. Immunostaining with specific antisera reveals predominantly nuclear accumulation of both proteins (Figure 2A). Although both constructs express well in COS cells, we consistently failed to detect the intact protein after overexpression of XTRPS1 in Xenopus embryos, which is important for subsequent experiments. Therefore, we constructed a truncated protein (XTRPS1ΔN) lacking the first 805 amino acids, which also localizes to the nucleus (Figure 2A). XTRPS1ΔN, which includes the solitary GATA-type zinc finger, binds specifically to the consensus GATA sequence in gel mobility shift assays (Figure 2B). This is confirmed by competition from oligonucleotides containing the sequence GATA but not from those containing the sequence GATC. Interaction with DNA is lost when the GATA-type zinc finger is disrupted, as in the mutant construct XTRPS1ΔNmut, or in the presence of a specific XTRPS1 antiserum. Thus, TRPS1 possesses defining properties of a GATA protein by virtue of its amino acid sequence and specific binding to DNA. Figure 2.TRPS1 is a sequence-specific, DNA-binding nuclear protein. (A) Subcellular localization by immunofluorescence of COS cells transfected with cDNA constructs encoding full-length XTRPS1 (top), an N-terminal truncated protein (XTRPS1ΔN, middle) or full-length mTRPS1 (bottom). Cells transfected with Xenopus or mouse proteins were stained with rabbit antisera directed against Xenopus and mouse peptides, respectively. Results with the appropriate pre-immune serum and with 4′-6-diamidine-2-phenylindole (DAPI) nuclear stain are also shown. (B) Electrophoretic mobility shift assay (EMSA) of in vitro translated XTRPS1ΔN using a double-stranded GATA oligonucleotide probe. Cold competitions are with either the same oligonucleotide or one in which the GATA sequence was altered to GATC. The complex formed with XTRPS1ΔN (arrow) is abrogated in the presence of appropriate antiserum. Full-length XGATA4, and a mutant protein in which two cysteine residues within the XTRPS1 GATA-type zinc finger are altered (XTRPS1ΔNmut), serve as additional controls. Download figure Download PowerPoint TRPS1 functions as a transcriptional repressor In many experiments with several variations, XTRPS1 and mTRPS1 failed to activate a luciferase reporter gene under the control of a GATA-dependent promoter (data not shown), whereas the representative vertebrate GATA protein XGATA4 consistently activated this reporter in mammalian cells (Figure 3A). We therefore tested the possibility that TRPS1 functions instead as a transcriptional repressor. Indeed, in co-transfection experiments, XTRPS1, XTRPS1ΔN and mTRPS1 potently repress the GATA-dependent activation induced by XGATA4 (Figure 3A). The degree of repression is proportional to the amount of co-transfected plasmid and, like the binding to DNA, also depends on integrity of the GATA-type zinc finger. The mutant proteins XTRPS1mut, XTRPS1ΔNmut and mTRPS1mut, in which two cysteine residues in this zinc finger are altered, fail to repress the transcriptional activation induced by XGATA4 (Figure 3A). Co-transfection of TRPS1 with TCF-1 and β-catenin does not repress the GATA-independent activation of a reporter gene regulated by the unrelated Tcf-family proteins (Figure 3B). Thus, repression by TRPS1 occurs specifically in the context of GATA cis-elements and represents a novel intrinsic activity for a vertebrate GATA factor. Figure 3.TRPS1 functions as a sequence-specific transcriptional repressor. Results of transient transfection of COS cells with a GATA-dependent luciferase reporter (A) and expression constructs encoding the transcriptional activator XGATA4 either alone or in combination with constructs expressing full-length XTRPS1, the truncated protein XTRPS1ΔN, full-length mTRPS1 and the corresponding mutant proteins XTRPS1mut, XTRPS1ΔNmut and mTRPS1mut, in which the GATA-type zinc finger is disrupted. Mut reporter, a reporter carrying mutated GATA sites. Dose dependence of XTRPS1-mediated transcriptional repression was established by co-transfection of XGATA4 and XTRPS1 plasmids in the indicated ratios. (B) TRPS1 fails to repress a GATA-independent reporter that is activated by the combination of a TCF-family protein and β-catenin. Download figure Download PowerPoint We and others have shown that some of the known GATA proteins induce endoderm differentiation in ectodermal explants of early Xenopus embryos (Shoichet et al., 2000; Weber et al., 2000). This activity is likely to reside downstream of the primitive endoderm inducer Mixer/Mix.3 (Henry and Melton, 1998; Mead et al., 1998) and provides an independent assay to verify the transcriptional repression function of TRPS1. We therefore co-expressed TRPS1 and XGATA4 mRNAs in Xenopus embryos and assayed ectodermal explants for ectopic expression of the endodermal markers intestinal (IFABP) and liver-specific (LFABP) fatty acid binding proteins and XGATA5 (Shi and Hayes, 1994; Henry and Melton, 1998; Mead et al., 1998). XTRPS1ΔN and mTRPS1 potently repress XGATA4-induced expression of each of these lineage markers (Figure 4A and B). This repression relies on an intact GATA-type zinc finger (data not shown) and is also seen when endoderm is induced by Mixer (Figure 4B). Results on representative samples from lanes 3 and 4 in Figure 4A confirm that the PCRs were performed in the linear range of amplification (Figure 4C). Thus, TRPS1 functions as a sequence-specific repressor of GATA-dependent processes in two independent biological assays. Figure 4.TRPS1 represses GATA-mediated induction of endoderm in Xenopus embryos. RT–PCR analysis of the endodermal marker genes IFABP, LFABP and XGATA5 on explanted Xenopus animal caps co-injected with 400 pg of XGATA4 (A and B) or Mixer (B) mRNA and 300–600 pg of XTRPS1ΔN or 600 pg of mTRPS1 mRNA. Controls include embryos injected with H2O or with a neutral filler (Ctl) RNA, and RT–PCR analysis for EF-1α and on samples not treated with reverse transcriptase (−RT). Results are representative of four independent experiments. (C) PCR on representative samples [lanes 3 and 4 from (A)] confirms that reactions were performed in the linear range of amplification. Download figure Download PowerPoint The repressive function of TRPS1 maps to the two C-terminal Ikaros-related zinc fingers Transcriptional repression by TRPS1 may occur through competition for binding to GATA sites in DNA or, alternatively, through an active mechanism that requires additional motifs. Repression by mTRPS1 and XTRPS1ΔN are comparable (Figures 3 and 4), which indicates that the first ∼800 amino acids of TRPS1 may be dispensable for this function. To map the repressive domain more precisely, we constructed two C-terminal deletions of 320 (XTRPS1ΔNΔC320) or 119 (XTRPS1ΔNΔC119) amino acids (Figure 5A, constructs 2 and 3). As expected, each resulting protein retains specific binding to the GATA consensus sequence (Figure 5B) but does not itself activate a GATA-dependent reporter (data not shown). Neither protein, including the shorter one which can bind DNA efficiently (construct 2), represses XGATA4-induced activation (Figure 5C). These results point to the requirement for a region contained within the terminal 119 residues. Figure 5.The repressor domain of TRPS1 maps to a C-terminal region encompassing two Ikaros-type zinc fingers. (A) Schematic of expression constructs 1–5 used in these experiments. 1–3 are truncation mutants of XTRPS1; 4 and 5 are fusion constructs between portions of the transcriptional activator XGATA4 and the C-terminal 119 residues of XTRPS1. G-Zn and C-Zn designate the TRPS1 GATA-type zinc finger and the C-terminal zinc finger of XGATA4, respectively. (B) EMSA analysis of the proteins encoded by constructs 1–5 using a GATA probe and competitor oligonucleotides as shown in Figure 2. (C) Results of transient transfection of COS cells with a GATA-dependent luciferase reporter and either constructs 4 or 5 alone, or constructs encoding XGATA4 either alone or in combination with constructs 1–5. Mut rep, a reporter carrying mutated GATA sites. (D) RT–PCR analysis of IFABP expression in explanted Xenopus animal caps injected with H2O or co-injected with XGATA4 (400 pg) and the indicated TRPS1 (600 pg) mRNAs. (E) Comparison of the amino acid sequence of mTRPS1 residues 1204–1270 with the C-terminus of each murine Ikaros-family protein. Identical amino acid residues are shaded in black and conservative substitutions in gray. Download figure Download PowerPoint To verify these results independently, we co-expressed XGATA4 and truncated XTRPS1 mRNAs in Xenopus animal caps and examined ectopic endoderm differentiation. Again, neither XTRPS1ΔNΔC119 nor the mutant protein XTRPS1ΔNmut (in which the GATA-type zinc finger is disrupted) represses XGATA4-induced expression of IFABP (Figure 5D). These findings confirm that the repressive function of TRPS1 requires a C-terminal region of, at most, 119 amino acids. This region encompasses a double zinc finger motif found in the Ikaros family of transcriptional regulators (Georgopoulos et al., 1997). mTRPS1 shows 45–50% sequence similarity with other Ikaros-type zinc fingers in this domain, over which all the known Ikaros family proteins share >80% similarity with each other (Figure 5E). TRPS1 is the only vertebrate or invertebrate protein known to possess both GATA- and Ikaros-type zinc fingers. To determine whether this region functions as an autonomous repression module, we created two fusion constructs between the activator XGATA4 and the C-terminal 119 residues of XTRPS1 (Figure 5A, constructs 4 and 5). XGATA4+C119 retains almost the complete sequence of XGATA4, while the control (XGATA4tr+C119) is truncated in the basic region adjacent to the GATA-type zinc finger and so does not bind DNA (Figure 5B). The fusion protein XGATA4+C119 represses GATA4-induced activation to basal levels, whereas XGATA4tr+C119 does not (Figure 5C). Thus, the transcriptional repression activity of TRPS1 is imparted by a conserved C-terminal domain that can override the intrinsic activation capacity of a typical GATA protein such as GATA4. Developmental expression of mTRPS1 Patients with TRPS types I and III have short stature, hip and phalangeal malformations, characteristic facial anomalies and sparse hair (Langer et al., 1984). However, it is not known whether the disease results from TRPS1 expression in the affected tissues or in other sites. Skeletal malformations are manifested much after birth and are progressive, but it is not known whether TRPS1 expression is important principally during development or throughout life. In mouse embryos, TRPS1 mRNA is detected prior to E7.5, with peak levels at around E11.5, and expression in mid-gestation is detected in both visceral and skeletal tissues (data not shown). Although these findings raise the possibility that transient TRPS1 activity may modulate many developmental processes, the highest TRPS1 expression in mid-gestation correlates precisely with sites of documented involvement in the TRP syndromes. mRNA in situ hybridization analysis reveals intense TRPS1 expression in the maxilla, mandible and snout at E12.5 (Figure 6A); lateral expression is higher than in the midline (Figure 6B). Expression in visceral organs, detected readily by northern analysis (data not shown), is not revealed by in situ hybridization, suggesting that mRNA expression in the developing face is especially high. At E12.5 and E13.5, prominent expression is also observed in the prospective phalanges (Figure 6C) and in the femoral head within the developing hip (Figure 6D). The only other site of high TRPS1 mRNA expression is in the hair follicles (Figure 6E). Northern analysis reveals that TRPS1 mRNA levels in the developing limbs and face are highest at E13.5 and decline dramatically thereafter (Figure 6F). These observations strongly suggest that the cardinal features of TRPS result from defects that occur during formative stages in the fetal development of tissues with high TRPS1 expression. Figure 6.Correlations between TRPS1 in development and disease. (A–E) mRNA in situ hybridization analysis of mTRPS1 expression in whole mouse embryos (A), the jaw (B, coronal section), digits (C), femoral head (D) and scalp hair follicles (E) at E12.5–13.5. Hybridization with a sense probe yielded no detectable signal (data not shown). (F) Northern analysis of mTRPS1 (T) or GAPDH (G) expression in tissues isolated from mouse fetuses at the indicated gestational age. FL, forelimbs; HL, hind limbs. (G) Map from the Jackson Laboratory BSS backcross showing a portion of chromosome 15 with loci linked to Trps1, depicted with the centromere at the top. Percentage recombination between adjacent loci (±SE) are indicated to the left and gene symbols to the right. Loci mapping to the same position are listed on the same line. Missing typings were inferred from surrounding data where assignment was unambiguous. Panel data and references for mapping other depicted loci are available at http://www.jax.org/resources/documents/cmdata. (H) FISH analysis of hTRPS1 showing localization to chromosome 8q23–24. Download figure Download PowerPoint We mapped mTrps1 by Southern analysis using an interspecific backcross panel (Rowe et al., 1994). mTrps1 is non-recombinant with D15Mit143, D15Wsu126 and Atox1, placing it on mouse chromosome 15, 28 cM distal to the centromere (Figure 6G). This region shows extensive conservation with human chromosome 8q, and also a small region of conservation with human 5q (http://www.informatics.jax.org). We confirmed the chromosomal location of the human TRPS1 locus by fluorescent in situ hybridization (FISH). The specific probe hybridized to chromosome 8q23–8q24 on metaphase spreads of normal human lymphocytes (Figure 6H, red signal); an alpha-satellite probe specific for chromosome 8 (green signal) is shown as a control. These results are consistent with those from positional cloning of hTRPS1 (Momeni et al., 2000) and with the high conservation between the murine and human TRPS1 genes (Figure 1). Mutations in TRPS kindreds encode truncated proteins that antagonize TRPS1 function Among the six mutations identified originally in families with type I TRPS, five result in truncation of the protein N-terminal to the GATA-type zinc finger and one leads to truncation between the GATA- and Ikaros-type zinc fingers (Momeni et al., 2000). All patients retain one wild-type allele, which has been interpreted to suggest that TRPS is a haploinsufficiency syndrome. Our findings raise the alternative possibility that truncated proteins lacking the GATA- and/or Ikaros-type zinc fingers function as dominant inhibitors of the wild-type protein. To test this possibility, we co-expressed four disease forms of mTRPS1 (P1–P4) together with the wild-type cDNA in each of the available assays for its transcriptional repression function. TRPS1-mediated repression in transfected cells is partially relieved upon co-expression of each of the four truncation mutants we examined (Figure 7A, compare lanes 2, 3 and 4–7); none of these mutants shows independent transactivation (lanes 8–11) or repression (data not shown). We also co-expressed XGATA4 and mTRPS1 in Xenopus animal caps together with mRNAs encoding two of the truncated forms. The repressive activity of mTRPS1 is completely abrogated by both P1 and P3, as shown by rescued expression of all three endodermal markers tested (Figure 7B, compare lanes 6 and 4–5). P1 and P3 lack intrinsic repressive activity (lanes 1–3), as predicted. This result does not reflect a trivial effect on the level of injected mTRPS1 mRNA, which is detected at the same level in the presence or absence of the disease isoforms P1 and P3 (Figure 7B, bottom panel; the PCR primers used here detect full-length mTRPS1 but not P1 or P3). Our findings hence suggest that the reason for autosomal dominant inheritance of type I TRPS may be that the function of the wild-type allele is effectively antagonized by the truncated proteins. Figure 7.TRPS1 truncation mutations associated with type I tricho-rhino-phalangeal syndrome function as dominant inhibitors of the wild-type protein. (A) Results of transient transfection of
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