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  A number of transcription factors are involved in the cellular transitions that take place in embryonic nerves and early post-natal nerves ( 9, 10 ). The HMG domain factor Sox-10 is expressed in most or all neural crest cells and is required for th...

  Eight zinc finger proteins, five of which have been shown to be transcription factors, have previously been identified in Schwann cells ( 13, 19, 22 – 25 ). Three of the transcription factors are members of the Egr family, which has four members. T...

  The Egr family, however, comprises only a small proportion of the total number of known zinc finger transcription factors, more than 700 of which have been identified. Among the zinc finger genes about one third contain an N-terminal domain known a...

  To isolate zinc finger genes that are expressed early in the Schwann cell lineage, we used RNA taken from embryonic peripheral nerves at a stage when Schwann cell precursors are the predominant cell population. We designed a screening strategy base...

        EXPERIMENTAL PROCEDURES       TOP   ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES   RESULTS   DISCUSSION   REFERENCES     Isolation of a Zinc Finger Motif-enriched cDNA Library— Total RNA was prepared from freshly dissected tissues of E12...

  Randomly selected clones of the zinc finger-enriched cDNA library were grown as single colonies on LB agar plates, transferred onto Hybond-N nylon membranes (Amersham Biosciences) and lysed. Duplicate filters were hybridized with cDNA probes prepar...

  Biostatistical analysis was performed using the programs BlastN, BlastP, and BlastX ( 40 ), LASERGENE (DNASTAR, Madison, WI), ExPasy ( www.expasy.ch ), SMART (smart.embl-heidelberg.de), Predict-protein ( www.embl-heidelberg.de/-predictprotein/ ) an...

   Cloning of the Zfp-57 Protein Coding Region— The full-length protein coding region of the rat Zfp-57 gene (amino acids 1–421) was obtained by RT-PCR using Pfu DNA polymerase (Stratagene, Inc., La Jolla, CA) and the primer pair ZFP57-USP2 (5'-GAATG...

   Cloning of the Full-length Mouse Zfp-57 Coding Sequence— The full-length mouse Zfp-57 coding sequence was cloned after reverse transcription of 0.5 µg of RNA isolated from E14.2 embryonic stem cells (a gift from Dr. Rosa Beddington), followed by P...

   Semiquantitative RT-PCR— In all cases RNA was extracted using TRIzol reagent. In experiments using embryonic, postnatal, and adult tissues first-strand cDNA was synthesized from total RNA as described above. The primers for semiquantitative RT-PCR...

  For the experiments with leukemia inhibitory factor (LIF, Merck Biosciences), cDNA was synthesized from 1 µg of RNA using random hexamers. GAPDH was used as an invariant control. The primers used for amplification were as follows: GAPDH-F: 5'-ACCAC...

  PCR conditions for the GAPDH RT-PCR were an initial step at 95 °C for 2 min, followed by 28 cycles at 94 °C for 30 s, 63 °C for 30 s, and 68 °C for 30 s, followed by a final extension step at 68 °C for 3 min. STAT3 and Zfp-57 RT-PCRs were carried o...

   Production of Recombinant ZFP-57 Protein in E. coli— Mouse Zfp-57 cDNA, representing nucleotides 16–1265 of the original protein coding sequence, was cloned into the prokaryotic expression vector pQE32 (Qiagen) to create an open reading frame cont...

   Nerve Transection and Crush— Adult male Wistar rats (180–220 g) were anesthetized and nerve cut and crush performed as described previously ( 44, 45 ). All experiments were in accordance with German and European Community guidelines on animal expe...

   Construction of Eukaryotic Expression and Transient Transfection Vectors— A PCR step was performed to replace amino acids 1–4 of mouse ZFP-57 with the synthetic primer ZFP57-USP3 (5'-GTGAAGCTTGGAAACAGTCTTCCCAGCCATCCAGGACAC-3') introducing a unique...

  To obtain green fluorescent protein (GFP) constructs for the nuclear localization experiments sequences coding for full-length ZFP-57 wild-type and mutant forms were amplified from a mouse Zfp-57 full-length cDNA clone using Pfu polymerase. Mutant ...

   

          View larger version (49K):  [in this window]  [in a new window]  F IG. 2. Biostatistical analysis of ZFP-57. A, alignment of mouse and rat ZFP-57 sequences. The top line indicates the mouse sequence and the middle line the rat sequence. As ...

       The PCR primers used to obtain the full-length Zfp-57 construct ( Zfp-57 wt-GFP) were F1 5'-GGAATTCGCCACCATGGCAGCTAGGAAACAGTCTTCC-3' and R1 5'-TTGGATCCGTCCGAATCTTCTTCTGTCAC-3'. Mutants Zfp-57 142–190 and Zfp-57 136–286 were obtained by amplify...

  For the transient transfections in COS-7 cells, DNA coding for the N-terminal sequence of ZFP-57 (amino acids 2–91) or for the C-terminal sequence lacking the KRAB domain 1–91 (amino acids 92–421) was cloned in-frame downstream from the GAL4 DBD (a...

  Plasmid constructs pX9.0MBPLuc, pXP 0 Luc, and pXPGKLuc containing 9.0 kb of the myelin basic protein ( MBP ) promoter and 1.1 kb of the P 0 promoter were generated as described previously and used in the transient transfection and promoter analysi...

   

   Cell Culture and Transient Transfections— For proliferation experiments Swiss 3T3 cells were grown in 10-cm tissue culture dishes (BD Biosciences) in high glucose (4500 mg/liter) Dulbecco's modified Eagle's medium (DMEM), 5% fetal calf serum (FCS)...

  To measure the response of Zfp-57 mRNA to an added growth factor, fresh culture medium was added to Swiss 3T3 cells 24 h before addition of fibroblast growth factor (FGF) 2 (12 ng/ml) in fresh culture medium. Total RNA was prepared from treated cul...

  For the nuclear localization experiments NIH3T3 cells were plated on glass coverslips, at 3000 cells in a 30-µl drop of DMEM/10% donor calf serum. Following attachment, the cells were cultured in 0.5 ml of the same medium and left for 24 h. They we...

  For the KRAB domain repression assays COS-7 cells were plated at a density of 1.4 x 10 6 cells in 10-cm dishes and transfected 48 h later, using a total of 2.6 µg of DNA per transfection, as described above. Cell lysates were collected 48 h later, ...

  For RNA extraction, primary cultures of astrocytes, oligodendrocytes, endothelial cells, pericytes, and neurons were a gift of Dr. Krause-Finkeldey. All cultures were prepared from newborn or perinatal rats as described previously ( 51 ). They were...

  For immunocytochemistry, promoter analysis, and to study the effect of LIF, Schwann cells were prepared from newborn or postnatal day (P) 3 or P4 rat sciatic nerve and serum-purified using cytosine arabinoside, followed in some experiments by antib...

  In transfection experiments with the 1.1-kb P 0 and 9.0-kb MBP promoters P3-purified Schwann cells were expanded by passaging three times in DMEM, 10% FCS, and 2 µ M forskolin (Merck Biosciences) and crude glial growth factor prepared from bovine p...

   Immunocytochemistry— Sciatic nerves from embryonic and newborn mice were teased on to gelatin coated microscope slides as described previously ( 57 ). Slides were air-dried for 30 min, fixed in ice-cold methanol for 10 min at 4 °C, then equilibrat...

        RESULTS       TOP   ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES   RESULTS   DISCUSSION   REFERENCES     Identification of ZFP-57, a Novel Zinc Finger Protein Expressed in the Schwann Cell Lineage— To isolate Schwann cell genes containin...

   

          View larger version (43K):  [in this window]  [in a new window]  F IG. 1. Outline of the strategy used to clone genes expressing zinc finger motif genes. A, diagram showing that zinc finger ( ZF ) motifs arranged in tandem arrays in a hypot...

       Data base analyses revealed that one sequence obtained was more than 95% identical to the murine transcription factor ZFP-57 previously identified by Okazaki et al. ( 61 ) in F9 teratocarcinoma cells. We cloned and sequenced over 11 kb of mous...

   Expression of Zfp-57 during Embryonic and Postnatal Development of the Sciatic Nerve— The expression of the Zfp-57 gene during the early and postnatal development of the mouse sciatic nerve was compared with the expression of the zinc finger trans...

   

          View larger version (65K):  [in this window]  [in a new window]  F IG. 3. ZFP-57 gene and protein expression during embryonic and postnatal development of mouse sciatic nerve. A, semiquantitative RT-PCR analysis using primers specific for Z...

       A polyclonal antibody that was raised against full-length histidine-tagged ZFP-57 fusion protein recognized the full-length ZFP-57 protein in lysates from IPTG induced bacteria using Western blot detection as shown in Fig. 3 D. By immuno-histo...

  Because many Schwann cell associated genes are axonally controlled ( 7 ), we tested whether Zfp-57 mRNA expression in Schwann cells was affected by loss of axonal contact in vivo, which results in re-entry of Schwann cells to the cell cycle and los...

   Zfp-57 mRNA Is Differentially Expressed in Neural Tissues during Development— We next compared Zfp-57 expression in different parts of the nervous system. Steady-state mRNA expression levels of Zfp-57 were determined at embryonic (E12 and E15) and...

   

          View larger version (33K):  [in this window]  [in a new window]  F IG. 4. Tissue-specific expression of Zfp-57 in the mouse. A, RT-PCR analysis of total RNA extracted from freshly dissected tissues of embryonic ( E ) or newborn mice as show...

       We further examined Zfp-57 expression in the adult murine nervous system ( Fig. 4 B ). As expected, when compared with the earlier time points, mRNA levels were lower. To compensate for this reduced expression, the number of amplification cycl...

  Next, the expression of Zfp-57 in cultured neural cells was examined ( Fig. 4 C ). Total RNA was isolated from primary cultures of neurons, oligodendrocytes, astrocytes, endothelial cells, and pericytes, all of which are derived from early postnata...

   The Subcellular Localization of ZFP-57— To analyze the subcellular localization of ZFP-57 protein a transient transfection protocol was established. For these experiments we used NIH3T3 cells to allow easy visualization of both the cytoplasmic and...

   

          View larger version (37K):  [in this window]  [in a new window]  F IG. 5. ZFP-57 protein localizes to heterochromatin-rich nuclear areas. A, left hand panels show the EGFP fluorescence revealing the localization of the ZFP-57 wild-type and ...

       To investigate the endogenous distribution of ZFP-57 and HP1 in Schwann cells we double-labeled neonatal mouse Schwann cells cultured in neuregulin with antibodies to ZFP-57 and HP1. Substantial co-localization of the two proteins was seen wit...

   Transcriptional Repression Activity of the ZFP-57 KRAB Domain— KRAB domains silence both basal and activated transcription in transfected cells ( 28, 30, 62 ). We therefore tested whether the KRAB sequence in ZFP-57 could down-regulate VP16-ER DBD...

   

          View larger version (21K):  [in this window]  [in a new window]  F IG. 6. A, the ZFP-57 KRAB domain represses VP16-ER DBD-mediated activation of a GAL4-ERE-tk-CAT reporter construct. COS-7 cells were transfected with 0.5 µg of a GAL4 DBD ex...

       The DNA sequence coding for the N terminus of the ZFP-57 protein, down to the first zinc finger, was fused to GAL4 DBD in the vector pBXG1. The Zfp-57 start codon was eliminated so that a fusion protein was obtained where the DBD of GAL4 (amin...

   ZFP-57 Suppresses Promoter Activity in Co-transfection Experiments— To test whether ZFP-57 represssed identified genes, including myelin genes, we co-transfected Schwann cells with Zfp-57 and each of the following three promoters: 1.1 kb of the P ...

   

          View larger version (18K):  [in this window]  [in a new window]  F IG. 7. ZFP-57 represses gene promoter activity. A, cells were co-transfected with Zfp-57 in the sense ( ZFP-57 ) or antisense ( ZFP-57 AS ) orientation or with empty cytomeg...

        LIF Up-regulates Zfp-57 Expression via the JAK/STAT Pathway— ZFP-57 is expressed in F9 teratocarcinoma cells, and down-regulated upon differentiation of these cells ( 61 ), and we find that it is also expressed by embryonic stem cells (not sh...

  RNA was isolated at different time points (30 min and 1, 3, and 6 h) after addition of LIF (20 ng/ml) and the levels of Zfp-57 transcript established via semiquantitative RT-PCR, following normalization to GAPDH. RNA was also isolated at the 1-h po...

  We found that Zfp-57 mRNA was up-regulated 1 h after addition of LIF. These levels then decreased to control levels by the 6-h time point. The AG490 inhibitor abolished LIF-induced up-regulation of Zfp-57 ( Fig. 8 ). This suggests that LIF activate...

   

          View larger version (28K):  [in this window]  [in a new window]  F IG. 8. Up-regulation of Zfp-57 transcripts following stimulation of Schwann cell cultures with LIF. Semiquantitative RT-PCRs of Zfp-57, STAT3, and GAPDH, on cDNA prepared fr...

        Zfp-57 and Proliferation— The absence of a significant change in Zfp-57 mRNA levels in cut nerves, when Schwann cells transit from quiescence to proliferation, suggested that the gene was not strongly associated with cell division in Schwann ...

  The percentage of BrdUrd-positive cells was 21.0 ± 3.6% in control cells with empty vector, 17.0 ± 10% in control cells with Zfp-57 in the antisense direction, and 27.3 ± 3.9% in control cells with Zfp-57 in the sense direction, indicating that ZFP...

   

          View larger version (52K):  [in this window]  [in a new window]  F IG. 9. Expression of Zfp-57 and Krox-20 in Swiss 3T3 cells in the presence of FGF-2. Semiquantitative RT-PCR analysis of cultured Swiss 3T3 cells at different time points af...

             DISCUSSION       TOP   ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES   RESULTS   DISCUSSION   REFERENCES    We have isolated the rat Zfp-57 gene using a fast and simple approach to search for novel zinc finger transcription factors e...

  ZFP-57 is a relative of the Xenopus laevis transcription factor IIIA type gene family of zinc finger transcription factors ( 66 ). In addition to the C2H2 zinc finger motif ( 67 ) it also contains a KRAB-DNA binding motif, a characteristic shared b...

  In other zinc finger proteins the KRAB motif is functionally associated with transcriptional repression ( 62, 71 ). Consistent with this, we find that the KRAB domain in ZFP-57 acts as a powerful repressor of activated transcription when linked to ...

  The KRAB domain is also necessary for the normal localization of the protein to speckles of centromeric heterochromatin within the nucleus of NIH3T3 cells, another characteristic typical of proteins containing this domain ( 36, 62, 71 ). Neverthele...

  Consistent with the cloning of Zfp-57 from E12 sciatic nerve, mRNA expression was relatively high during early development of peripheral nerves. We also found relatively high levels of expression in spinal cord and sensory ganglia at E12, with high...

   

  In peripheral nerve, Zfp-57 mRNA is substantially down-regulated after a peak of expression between E15 and E17 and by P28 has dropped to low levels, though the protein is still readily detectable in adult nerve. The onset of down-regulation overla...

  In line with this, we tested the hypothesis that ZFP-57 function might be associated with cell proliferation using a variety of conditions. In Swiss 3T3 cells the number of cells synthesizing DNA is not altered by enforced expression of ZFP-57 (see...

  Despite the fact that ZFP-57 can repress myelin gene promoters when constitutively expressed in Schwann cells, it does not inhibit induction of Krox-20 or the myelin proteins periaxin or P 0 in a cAMP-based assay of myelin-related differentiation (...

  In conclusion, this is the first report of a zinc finger protein containing a KRAB domain with a demonstrated function as a transcriptional repressor in Schwann cells of the peripheral nervous system. Zfp-57 is under strong developmental regulation...

         FOOTNOTES       The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AY344233  [GenBank]. 

    * This work was supported in part by a grant (to K. R. J. and R. M.) from the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked " advertisem...

   Both authors contributed equally to this work.   

   Current address: Oncology Disease Group, Aventis Pharma, 13 Quai Jules Guesde, 94403 Vitry-sur-Seine, France.   

   Current address: Howard Hughes Medical Institute and Developmental Biology Program, Memorial Sloan-Kettering Center, New York, NY 10021.   

    var u = "r.mirsky", d = "ucl.ac.uk"; document.getElementById("em0").innerHTML = ' ' + u + '@' + d + '<\/a>'//-->. 

   1 The abbreviations used are: E, embryo day; Zfp, zinc finger protein; KRAB, Krüppel-associated box; DBD, DNA binding domain; HP1, heterochromatin protein 1; LIF, leukemia inhibitory factor; GFP, green fluorescent protein; CAT, chloramphenicol ace...

   2 C. Taveggia, A. Pizzagalli, E. Fagiani, M. L. Feltri, R. Forghani, A. Messing, A. C. Peterson, and L. Wrabetz, submitted manuscript.   

   3 Cited by T. Akagi, M. Usada, S. A. Jaradat, M. Ko, H. Niwa, and T. Yokota at 157.82.98.20/imswww/KenkyuGaiyou/AnnualReport2002/122-148.pdf.   

         ACKNOWLEDGMENTS    We thank Dr. Mark Ptashne (Memorial Sloan-Kettering Cancer Center, New York) for the pBXG1 construct, Dr. Masa Tada (UCL) for help and comments, Professor Pierre Chambon (IGBMC, Université Louis Pasteur) for the pG4MpolyII...

        REFERENCES       TOP   ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES   RESULTS   DISCUSSION   REFERENCES       Jessen, K. R., Brennan, A., Morgan, L., Mirsky, R., Kent, A., Hashimoto, Y., and Gavrilovic, J. (1994) Neuron 12, 509-527 [Cros...

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