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Previous Article | Next Article 
Molecular and Cellular Biology, November 2002, p. 7633-7644, Vol. 22, No. 21
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.21.7633-7644.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232
Received 27 March 2002/ Returned for modification 22 May 2002/ Accepted 8 August 2002
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ABSTRACT |
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INTRODUCTION |
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Three classes of Smad proteins—receptor-activated Smads (R-Smads), common Smads (co-Smads), and inhibitory Smads (I-Smads)—have been identified in mammals. Smads 1, 5, and 8 are R-Smads that primarily mediate BMP signaling, while Smads 2, 3, are mainly involved in mediating TGF-ß and activin signaling. Upon phosphorylation by type I receptors, R-Smads oligomerize with the co-Smad Smad4, which is essential for the BMP, TGF-ß, and activin signaling pathways. The complex translocates to the nucleus and interacts with other cofactors to regulate the expression of downstream target genes. Smad6 and Smad7 are I-Smads that can interfere with R-Smad/receptor or R-Smad/co-Smad complex formation to inhibit BMP/TGF-ß signaling pathways.
An intriguing question is how the relatively simple group of R-Smads mediates the many complex biological processes triggered in different tissues by BMP/TGF-ß signaling. Accumulated evidence suggests that the different responses of cells to BMP/TGF-ß ligands are mediated by interaction of R-Smads with cell-specific or stage-specific cofactors (1, 2, 38). Many nuclear proteins such as c-Jun/c-Fos (48), SnoN (31), Ski (23, 32), TGF-ß induced factor (TGIF) (43), Sp1 (7), Lef1/Tcf1 (18), and GATA3 (5) have been identified as TGF-ß Smad interaction partners. By contrast, relatively few BMP Smad interaction partners have been found.
It has been reported that Smad1 can interact with Hoxc-8 to dislodge it from its binding target, resulting in the activation of target genes (29). In another example, the zinc finger factor OAZ, originally identified as a nuclear partner of Olf-1/EBF, involved in olfactory epithelium and lymphocyte development, activates the homeobox gene Xvent-2 with a different group of zinc fingers than those for the Olf signaling pathway (10). Two negative transcriptional regulators of BMP Smads, Ski and Tob, have been identified recently (40, 46). We speculated that there are more transcription factors that can interact with BMP Smads, and identification of these Smad interaction partners is essential for elucidating the molecular mechanism for the various activities of BMPs during development.
Previous work from our laboratory has established that BMP signaling plays critical roles during male germ cell development. Bmp8b is required for the survival of germ cells in adult male mice, while Bmp8a is required for maintaining spermatogenesis and the integrity of the epididymis (50, 52). More recently, Zhao et al. (49) showed that mutation of Bmp7 enhances the defects in spermatogenesis caused by mutation of Bmp8a, further emphasizing the critical roles of BMPs during spermatogenesis. Smad1, a BMP-regulated R-Smad, is transcribed in the testes with a stage- and germ cell-specific pattern that is in contrast to its ubiquitous expression in mouse embryos (51). The molecular mechanism of BMP signaling during spermatogenesis is unknown.
In this paper, we describe the isolation of a mouse Krüppel-type zinc finger protein, mZnf8, as a novel interaction partner of Smad1 from a yeast two-hybrid screen. The interaction between mZnf8 and Smad1 was further confirmed with other experimental approaches. Functional analysis suggests that mZnf8 is a nuclear transcriptional repressor. Significantly, this gene is highly expressed in adult mouse testes, where it has an expression pattern similar to that of Smad1, as determined by section in situ hybridization. We propose that mZnf8 plays important roles in mediating BMP signaling during spermatogenesis.
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MATERIALS AND METHODS |
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The Smad1 bait plasmid was used to screen a mouse embryo (day of embryonic development 9.5 [E9.5] and E10.5) two-hybrid library (kindly provided by Stanley Hollenberg, Fred Hutchinson Cancer Research Center) with the Matchmaker Two-Hybrid System 3 (Clontech) following the manufacturer's instructions. Plasmids from positive clones were purified and cotransformed with the Smad2 bait construct into AH109 yeast cells to identify candidates that specifically interact with Smad1 but not with Smad2. Growth on selective medium (lacking Trp, Leu, His, and Ade) indicates positive interaction between bait and prey (Clontech). Two clones, S1 and S2, were identified from the screening. To perform the reciprocal two-hybrid assay, the insert from S1 was cloned into the bait vector pGBKT7, which was then cotransformed with different prey plasmids (as indicated in Fig. 1) into AH109 cells.
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Cloning full-length mZnf8. The insert of clone S1, which encodes a partial cDNA sequence highly homologous to human ZNF8 (20), was used as a probe to screen an E8.5 to E9.0 mouse embryonic cDNA library (Stratagene) at high stringency as described before (51). Three positive clones were obtained out of 5 x 106 plaques. Sequencing analysis indicated that they were all identical clones containing the 5' untranslated region and the coding sequence of the N-terminal part of mZnf8 (see below). The 5'-most ATG gives the longest reading frame and is surrounded by a Kozak consensus sequence. An in-frame stop codon is present upstream of the first ATG. Thus, the first ATG is probably the authentic start codon of mZnf8. No in-frame stop codon was identified in the 3' end of three clones.
To acquire a full-length mZnf8 transcript, 3' rapid amplification of cDNA ends (RACE) was performed with Marathon-Ready cDNA (Clontech) following the manufacturer's instructions. A single DNA fragment was acquired and then sequenced. The putative full-length mZnf8 mRNA was assembled with the clones obtained from the cDNA library screening and the PCR product from 3' RACE. The cDNA sequence of mZnf8 was submitted to GenBank under accession no. AF480861.
In vitro GST pulldown assay. Full-length Smad1, Smad2, and Smad5 coding sequences or MH1 (amino acids 1 to 145), Linker (amino acids 146 to 266), and MH2 (amino acids 267 to 465) domains of Smad1 were PCR amplified and cloned into the BamHI site of pGEX-2T. The glutathione S-transferase (GST)-Smad3 and GST-Smad4 fusion constructs were gifts of X. Cao (University of Alabama) and R. Derynck (University of California at San Francisco), respectively. GST or GST fusion proteins were purified with glutathione-conjugated agarose beads (Pharmacia) from BL21 following the manufacturer's instruction. Full-length mZnf8 was labeled with [35S]methionine with the TNT coupled transcription system (Promega) according to the manufacturer's protocol. GST pulldown assays were performed as described before (26). Samples were separated via reducing sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (SDS-PAGE). Gels were first stained with Coomassie blue to visualize GST fusion proteins and then treated with Enhance (NEN) following the manufacturer's instructions. The treated gels were dried before autoradiography.
To quantify the probes pulled down with GST fusion proteins, bands corresponding to the mZnf8 probe were excised, and radioactivity was measured in a scintillation counter (Beckman LS3801).
Copurification of GST-mZnf8 and HA-Smad proteins from mammalian cells. The full-length mZnf8 coding sequence was PCR amplified and cloned into the BamHI site of pCMV-GST (35), a eukaryotic GST expression vector. Smad3 or Smad4 coding sequence was PCR amplified and cloned into the pCMV-HA vector (Clontech). The MH1, Linker, and MH2 domains of Smad1 were removed from bacterial GST expression constructs (described above) and cloned into the BamHI site of the pGCN-HA vector (45). The GST-mZnf8 fusion construct was cotransfected with different hemagglutinin (HA)-tagged Smad constructs (as indicated in the figures) into COS-M6 cells (gift of L. Limbird, Vanderbilt University Medical Center) with the Fugene 6 transfection reagent (Roche). ALK6 (Q203D) or ALK5 (T204D) constructs encoding a constitutively active form of BMPRIB or TGF-ßRI (gifts of L. Attisano, University of Toronto) were cotransfected as needed.
Purification of GST protein or GST-mZnf8 fusion protein was performed as described previously (35). Monoclonal GST (GST-2; Sigma) or HA (HA.11; Babco) antibodies were used to detect GST fusion proteins and HA-tagged Smad proteins, respectively. A horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) (Jackson) was used as the secondary antibody in Western analysis. A rabbit polyclonal anti-phospho-Smad1 serum (Upstate Biotechnology) and a horseradish peroxidase-conjugated anti-rabbit IgG (Jackson) were used to test whether the active form of Smad1 copurified with GST-mZnf8 from COS cells.
Cellular localization of mZnf8. The full coding sequence of mZnf8 was PCR amplified and cloned into the BamHI site of pCGN-HA (45). This construct was transfected into COS-M6 cells as above. At 36 h after transfection, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 15 min. Then 0.5 µM propidium iodide (Molecular Probes) was used to stain the nuclei following the manufacturer's instructions. A rat monoclonal HA antibody (3F10; Roche) and Cy2-conjugated anti-rat immunoglobulin secondary antibody (Jackson) were used to stain the fusion protein. Cells were observed with a fluorescent microscope.
Transient-transfection and luciferase reporter assays. In the mammalian two-hybrid assay, full-length Smad1 coding sequence was PCR amplified and cloned into the BamHI site of pFA-CMV (Stratagene) to acquire the GAL4 DNA binding domain (DB)-Smad1 fusion construct. To obtain the Znf8-vp16 construct, the potential Krüppel-associated box (KRAB; amino acids 25 to 96) coding region from pCGN-HA-mZnf8 (described above) was replaced with a sequence encoding the transcriptional activation domain of herpes simplex virus VP16. In the (Gal)5-E1b-lux reporter construct (gift of J. Massague, Memorial Sloan-Kettering Cancer Center), the transcription of the luciferase gene is driven by five copies of GAL4 DNA binding sites upstream of a basic E1b TATA box (43). Various constructs (as indicated in Fig. 4) were cotransfected into HEK293 cells; 0.2 µg of each plasmid was used for transfection unless otherwise indicated. The total amount of DNA used in all transfection assays was equalized by using pcDNA3 (Stratagene). Luciferase assays were performed with a dual luciferase reporter assay system (Promega) according to the manufacturer's protocol. pRL-TK (0.02 µg; Promega), which expresses Renilla luciferase (different from firefly luciferase) from a constant promoter, was cotransfected into HEK293 cells to normalize transfection efficiency in all assays.
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Constructs 12(GCCG)-lux (gift of K. Miyazono, Cancer Institute of the Japanese Foundation for Cancer Research), pTlx-lux (gift of J. Wrana, University of Toronto), and 3TP-lux (gift of J. Massague) were used to determine the effect of overexpression of mZnf8 on BMP and TGF-ß reporters.
To stimulate P19 cells with the BMP4 ligand, cells were treated with 100 ng of hBMP4 (purchased from R&D Systems, Inc.) per ml or with bovine serum albumin alone in Dulbecco's modified Eagle's medium with 0.1% fetal bovine serum for 14 to 16 h. Luciferase assays were then performed as described above. The Flag-Smad1 and Flag-Smad2 constructs were gifts of L. Attisano and J. Massague, respectively. Western analysis confirms that expression of Flag-Smad proteins was not decreased by increasing the amount of mZnf8 in transfections (data not shown). The pM3VP16 (Clontech) construct encoding the GAL4DB-VP16 fusion protein was used in a control experiment to exclude that mZnf8 functions as a general transcriptional repressor.
RT-PCR and RNA interference (RNAi) assays. Total RNA was isolated from cells with the RNeasy mini kit (Qiagen); 0.5 µg of total RNA was then subjected to reverse transcription (RT)-PCR analysis with the ThermoScript RT-PCR system (Life Technologies). Oligo(dT)20 instead of random hexamer was used for RT reactions. The expected PCR products for mZnf8 and ß-actin (positive control for RT-PCR) are 380 and 490 bp, respectively.
Synthetic oligonucleotides Primer2top (TTTGAGTCTTGAGCCCCGTCTACACGGTGTACGTGTAGACGGGGCTCAAGACTTTTTT) and Primer2bot (CTAGAAAAAAGTCTTGAGCCCCGTCTACACGTACACCGTGTAGACGGGGCTCAAGACT) were obtained from Integrated DNA Technologies, Inc. The two cDNA oligonucleotides were annealed and ligated into the vector mU6pro (gift of D. Turner, University of Michigan) to generate mU6pro-znf8, as described before (47). In mU6pro-znf8, a single short hairpin RNA corresponding to bp 1504 to 1529 of the mZnf8 coding region is transcribed from the U6 promoter. A total of 0.2 µg of reporter constructs with 1.0 µg of mU6proznf8 or 1.0 µg of mU6pro (vector alone control) were cotransfected into P19 cells, and luciferase assays were performed as described above.
Northern analysis. Northern blots containing mouse mRNA from different embryonic stages or from different adult mouse tissues were purchased from Clontech. Northern analysis was performed with ExpressHyb (Clontech) according to the manufacturer's instructions and with the full-length mZnf8 coding sequence as the probe. The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe (Clontech) was used for an mRNA loading control.
Section in situ hybridization. The testes from an 8-week-old ICR male mouse and mouse embryos were sectioned at 5 µm as described before (51). The insert from clone S1 was cloned into pBluescript (Stratagene) for making sense and antisense probes. The Smad1 probe and in situ hybridization assay were described before (51).
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RESULTS |
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To identify genes that are specifically involved in the BMP signaling pathway but not in the TGF-ß or activin pathway, plasmids from positive clones obtained from the first-round screen were retested for their ability to interact with Smad2 (see Materials and Methods). Two clones, S1 (identified seven times) and S2 (identified twice), encoded proteins that interacted only with Smad1 but not with Smad2 in yeast cells. This paper focuses on the characterization of clone S1.
As shown in Fig. 1A, transformants containing both S1 (in the prey construct) and the Smad1 bait construct grew on selective medium deficient in Leu, Trp, Ade, and His, indicating positive interaction between prey and bait. Positive interaction was not observed between clone S1 and Smad2, empty bait vector, or an unrelated protein. To further confirm the specific interaction between clone S1 and Smad1 in yeast cells, a reciprocal two-hybrid assay was performed (Fig. 1B). As in the forward two-hybrid assay, the insert of clone S1 in a bait construct could still interact with the Smad1 prey but not with the empty prey vector, Smad2, or an unrelated protein.
Sequencing analysis indicated that clone S1 contained a partial cDNA sequence highly homologous to the zinc finger regions of human zinc finger protein 8 (hZNF8) (20). The complete coding sequence of the mouse gene was acquired by screening a mouse E9.0 embryo cDNA library with S1 as the probe and 3' RACE (see Materials and Methods). The sequence of this cDNA (GenBank accession no. AF480861) contains an open reading frame of 1,719 nucleotides encoding a 573-amino-acid conceptual protein of 64 kDa.
Sequence alignment of the predicted mouse protein and human ZNF8 was performed with the program ClustalW (1.81) (Fig. 2). The extremely high conservation between the two proteins (74% identity and 91% similarity) indicates that the mouse gene is a true homolog of hZnf8. It encodes a Krüppel-type zinc finger protein with one potential KRAB domain at its N-terminal end and seven C2H2 zinc fingers in its C-terminal half. The KRAB domain has a transferable transcriptional repression activity that is required for the function of many Krüppel-type zinc finger proteins (6). The first six zinc fingers of mZnf8 are classical Krüppel-type fingers, with a conserved TGEKP(Y/F)X interfinger spacer (28), while the seventh finger is separated from the sixth by a longer spacer. The KRAB and zinc finger domains (including spacer regions) are the most highly conserved regions between the mouse and human proteins, consistent with the idea that they are critical for the function of Znf8 proteins. Regions outside of these two domains are less conserved, and no other potential functional domain was identified in the protein.
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In the next step, we applied a biochemical approach to test the interaction between the two proteins in mammalian cells. GST-mZnf8 and HA-Smad1 were coexpressed in COS-M6 cells, and the GST-mZnf8 fusion protein was purified from the cell lysate with GST binding beads. An anti-HA antibody was used to test whether HA-Smad1 copurified with GST-mZnf8 (Fig. 4B). In the absence of ALK6 (Q203D), which encodes a constitutively active form of BMPRI, only a trace amount of HA-Smad1 could be detected (lane 9 of Fig. 4B), but this was dramatically increased upon cotransfection of ALK6 (Q203D) (lane 2 of Fig. 4B). No HA-Smad1 was detected in the negative control lane (lane 1 of Fig. 4B), although equal amounts of HA-Smad1 were expressed (comparing lanes 1 and 2 in the bottom panel of Fig. 4B).
This result demonstrates that mZnf8 and Smad1 interact in mammalian cells and that interaction between the two proteins is greatly enhanced by BMP signaling. Since Smad1 is phosphorylated by BMPRI upon BMP stimulation (12, 48), we further confirmed that active (phosphorylated) Smad1 copurified with GST-mZnf8 by Western analysis with an anti-phospho-Smad1-specific antibody in Fig. 4C. Consistent with the result obtained from the in vitro pulldown assay (Fig. 3), the MH1 and MH2 domains but not the Linker domain of Smad1 were able to interact with mZnf8 in COS cells (lane 3 to lane 8 of Fig. 4B).
Interaction of mZnf8 with various Smad proteins. The results from yeast two-hybrid assays suggested that mZnf8 specifically interacts with BMP Smads but not with TGF-ß Smads. Surprisingly, in the in vitro GST pulldown assay, all Smad proteins tested (Smads 1 to 5) interacted with the radiolabeled mZnf8 probe (Fig. 5A). However, quantitative analysis indicates that mZnf8 had a higher binding affinity for BMP Smads (Smad1 and Smad5) than for other Smads tested (Fig. 5B). Similar results were also obtained from the copurification experiment in mammalian cells (Fig. 5C); more Smad1 and Smad5 copurified with GST-mZnf8 from COS cells than Smad2, -3, and -4. The results from both in vitro and in vivo experiments were repeated three to five times.
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mZnf8 is a nuclear transcriptional repressor. Since mZnf8 is a Krüppel-type zinc finger protein and interacts with phosphorylated, nucleus-localized Smad1, we speculated that mZnf8 is a nuclear protein, although no classical nuclear localization signal was identified from sequence analysis. To test this hypothesis, mZnf8 was tagged with HA and expressed in COS-M6 cells. As shown in Fig. 6A, HA-mZnf8 was localized exclusively in the nucleus, and its nuclear localization was not altered by stimulation with either 50 ng of hBMP4 or 5 ng of TGF-ß1 per ml for 1 h (data not shown). The same results were also obtained in HEK293 cells (data not shown). The exclusive nuclear localization of mZnf8 may explain the low level of mZnf8/Smad1 complex formed in COS cells in the absence of BMP stimulation (see above).
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To examine the potential function of mZnf8 in modulating transcriptional responses induced by BMP and TGF-ß, BMP-responsive and TGF-ß-responsive reporters were used in transient-transfection assays (Fig. 6C, D, and E). 3TP-lux contains TGF-ß-responsive elements derived from the plasminogen activator inhibitor 1 and collagenase promoters (44). Kusanagi et al. (17) found that reporters with multiple copies of the GCCGnCGC motif (GCCG box) respond specifically to BMP stimulation but not to TGF-ß or activin. The GCCG box was originally derived from Decapentaplegic (Dpp)-responsive promoters in Drosophila melanogaster (17). Later this motif was identified in the mouse Smad6 promoter and shown to be necessary for the transcriptional induction of mouse Smad6 by BMP stimulation (13).
Transcription of the luciferase reporter in pTlx-lux is driven by the BMP-specific responsive enhancer derived from Tlx-2 (33). As shown in Fig. 6C and D, overexpression of mZnf8 repressed both the TGF-ß and BMP reporters in a dose-dependent fashion. This result is consistent with the idea that mZnf8 functions as a transcriptional repressor and interacts with Smad1 and Smad2 in mammalian cells. The fact that overexpression of mZnf8 had a more dramatic effect on a BMP reporter than on a TGF-ß reporter is consistent with the observation that mZnf8 had a higher binding affinity for BMP Smads than for TGF-ß Smads. The results in Fig. 6E indicate that overexpression of mZnf8 also repressed the expression of BMP reporters in BMP ligand-induced responses. In a control experiment, overexpression of mZnf8 had no effect on GAL4-VP16-mediated transcription (Fig. 6F), indicating that mZnf8 does not function as a general transcriptional repressor.
Involvement of endogenous mZnf8 in the BMP signaling pathway. To directly test the role of mZnf8 in the BMP signaling pathway, we decided to silence (knock down) the expression of endogenous mZnf8 with an RNAi approach. It was reported recently that short hairpin RNA molecules can efficiently target corresponding mRNAs for degradation in mammalian cells (24, 47). For these experiments, we used P19 teratocarcinoma cells, which express endogenous mZnf8 (Fig. 7A). We made the construct mU6pro-znf8, which transcribes short hairpin RNA corresponding to bp 1504 to 1529 of the mZnf8 coding region (see Materials and Methods). Since no mZnf8 antibody is available, we could not examine the effect of this construct on the expression of the endogenous protein directly.
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mZnf8 is ubiquitously transcribed in mouse embryos. To begin to understand the in vivo function of mZnf8, we examined its expression pattern by Northern and section in situ hybridization assays. Results from Northern analysis indicated that transcription of mZnf8 was developmentally regulated during mouse embryogenesis (Fig. 8A). At E7, a very low level of mZnf8 transcripts could be detected. The mZnf8 transcript was most abundant at E11, and its level declined at later stages (E15 and E17). Section in situ hybridization analysis showed that mZnf8 was ubiquitously and evenly expressed in an E11.5 mouse embryo (Fig. 9). A similar expression pattern was also observed in E9.5 and E10.5 embryos (data not shown). The ubiquitous embryonic expression pattern of mZnf8 is identical to that of Smad1 (51) (data not shown) and is consistent with the possibility that mZnf8 interacts with Smad1 to modulate BMP signaling in many tissues during mouse embryogenesis.
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To test this prediction, we performed in situ hybridization on adjacent sections of an 8-week-old ICR male mouse with an mZnf8 antisense probe and a Smad1 antisense probe (Fig. 10). Consistent with our previous report (51), Smad1 transcripts were detected in certain tubules with variable intensity in a sexually mature mouse testis when the steady-state cycling of the seminiferous epithelium was established (Fig. 10). Significantly, mZnf8 was transcribed in the same apparent population of cells and to the same apparent level as Smad1 (Fig. 10), indicating that mZnf8 has a similar stage- and cell-specific transcription pattern. This result supports the hypothesis that mZnf8 interacts with Smad1 at specific stages during spermatogenesis.
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DISCUSSION |
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There are three possible models for mZnf8 function. First, it may bind a specific DNA sequence(s) to repress the transcription of target genes (Fig. 11A). This model is consistent with the observation that most of the known Krüppel zinc finger proteins interact with specific cis-regulatory DNA elements (reviewed in references 6 and 42). Upon BMP stimulation, Smad1 is phosphorylated and translocated into the nucleus. The binding of Smad1 to mZnf8 dislodges it from its binding sites and thus allows target genes to be transcribed. This derepression mechanism was proposed for the interaction between Hoxc-8 and Smad1 (29).
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In mammalian cells, Snip1 preferentially interacts with Smad4. Since this common Smad4 is required for the BMP, TGF-ß, and activin pathways (15), Snip1 is unable to set distinct thresholds for different TGF-ß superfamily cytokines. Thus, this potential appears to be unique to mZnf8. It is particularly interesting that mZnf8 can interact with both the MH1 and MH2 domains of Smad1. The MH1 domain is generally believed to be responsible for binding DNA, while the MH2 domain interacts with other partner proteins (1, 3, 25). Thus, the inhibition of Smad1 activity by mZnf8 can be twofold. First, mZnf8 interacts with the MH1 domain of Smad1 to block its DNA binding activity. Second, mZnf8 binds to the MH2 domain to block its trans-activation activity. Further studies are required to test this hypothesis.
Third, mZnf8 may function as a corepressor of Smad1 to actively repress the transcription of BMP downstream genes (Fig. 11C). A similar mechanism was proposed for a Smad2 interaction partner, TGIF (43). It was proposed that upon TGF-ß stimulation, Smad2 can interact either with coactivators, forming a transcriptional active complex, or with TGIF, forming a transcriptional repressor complex (43). mZnf8 may function in a similar way to form a transcriptional repressor complex with Smad1 to repress target genes. In addition to TGIF, SnoN and Ski were also identified as corepressors of TGF-ß Smads (23, 31, 32). Ski and Tob are two other known corepressors of BMP Smads (40, 46). mZnf8 is the third candidate as a corepressor of BMP Smads. Further experiments are required to distinguish among these three models.
Applying the RNAi knocking-down technique, we showed that decreasing expression of endogenous mZnf8 significantly increased the activity of one BMP reporter [12(GCCG)-lux] but not the other (pTlx-lux) in response to hBMP4 (Fig. 7). A simple explanation for this is that endogenous mZnf8 is selectively involved in certain responses of BMP signaling but not others. However, as we noticed in Fig. 7D, transcription of endogenous mZnf8 was not completely eliminated; likely some mZnf8 is still expressed, though we do not have the mZnf8-specific antibody to test this directly. Thus, we cannot exclude the possibility that a low level of endogenous mZnf8 is sufficient to suppress the activity of pTlx-lux. Another possible explanation is that an unknown corepressor of BMP Smad functions redundantly with mZnf8 in the pTlx-lux reporter but not in the 12(GCCG)-lux assay.
Krüppel-type zinc finger proteins belong to a very large family; it has been estimated that there are close to 300 Krüppel-type zinc finger proteins in humans (6, 16). About one third of these zinc finger proteins also contain a KRAB domain at their N-terminal ends. So far, mZnf8 is the only member of the KRAB transcriptional repressor family that may interact with Smad1 in vivo. It will be of interest in the future to determine what properties of mZnf8 make it a Smad1 interaction partner.
One interesting feature of mZnf8 is that its transcriptional repression activity was only slightly affected by deletion of the KRAB domain (Fig. 6B). This is different from most, if not all, other Krüppel zinc finger proteins studied (19, 30, 36, 41). The isolation of clone S1, which encodes the first five Krüppel-type zinc fingers, from the yeast two-hybrid screen suggests that these Krüppel-type zinc fingers do not possess transcriptional repression activity. Sequence comparison between the mouse and human proteins indicates that the seventh zinc finger sequence and the last 41 amino acids are highly conserved (Fig. 2). We speculate that one or both of the regions harbor the transcriptional repression activity. This will be tested in future studies.
BMP signaling plays critical roles in male germ cell development; deletion of Bmp8a or Bmp8b causes severe defects in spermatogenesis, and mutation in Bmp7 enhances the mutant phenotype of Bmp8a (49, 50, 52). However, the underlying molecular mechanism for BMP signaling remains unknown. In this study, we show that mZnf8 is highly transcribed in the testes of adult mice, in contrast to its ubiquitous transcription in mouse embryos. Furthermore, mZnf8 has a cell- and stage-specific transcription pattern similar to that of Smad1, the transcripts of which are mainly detected in the midstage pachytene spermatocytes (from stage V to stage XII) during cycling of the seminiferous epithelium, with the peak at stage X (51). This result is consistent with the hypothesis that the two proteins interact with each other at specific stages during spermatogenesis.
Smad2, a TGF-ß Smad protein, is expressed in preleptotene to pachytene spermatocytes (39). The different expression patterns of Smad1 and Smad2 suggest that BMP and TGF-ß are involved in different aspects of spermatogenesis (39). The similar transcription pattern of mZnf8 and Smad1 suggests that mZnf8 mainly mediates BMP signaling but not TGF-ß signaling during spermatogenesis. This is consistent with the discovery that mZnf8 has a higher binding affinity for BMP Smads (Smads 1 and 5) than for TGF-ß Smads (Smads 2 and 3). Thus, mZnf8 is so far the only candidate gene for mediating BMP signaling during mouse spermatogenesis. Future conditional gene inactivation experiments will help determine the exact function of mZnf8 during male germ cell development.
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ACKNOWLEDGMENTS |
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K.J. is an Associate and B.L.M.H. is an Investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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