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 J. Biol. Chem., Vol. 279, Issue 24, 25653-25664, June 11, 2004                                         This Article          Abstract                                                                        Full Text (PDF)                                                                                All Versions of this Article:    279/24/25653   most recent        M400415200v1                                            Alert me when this article is cited                 Alert me if a correction is posted                                    Citation Map        Services         Email this article to a friend                            Similar articles in this journal           Similar articles in PubMed              Alert me to new issues of the journal                Download to citation manager                                                              Citing Articles         Citing Articles via HighWire                        Citing Articles via Google Scholar                             Google Scholar         Articles by Alonso, M. B. D.                                  Articles by Mirsky, R.                      Search for Related Content                          PubMed         PubMed Citation               Articles by Alonso, M. B. D.          Articles by Mirsky, R.                                                                                                                                                                                 Identification and Characterization of ZFP-57, a Novel Zinc Finger Transcription Factor in the Mammalian Peripheral Nervous System *     María B. Durán Alonso,  Georg Zoidl ¶,  Carla Taveggia||,  Frank Bosse**,  Christiane Zoidl¶,  Mary Rahman,  Eric Parmantier,  Charlotte H. Dean,  Brett S. Harris¶¶,  Lawrence Wrabetz||,  Hans Werner Müller**,  Kristjan R. Jessen, and  Rhona Mirsky ||||  

 From the  Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom, the ¶ Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum, University Street, 44780 Bochum, Germany, the || San Raffaele Scientific Institute, DIBIT, Via Olgettina 58, 20132 Milan, Italy, the ** Molecular Neurobiology Laboratory, Department of Neurology, Heinrich-Heine-University, Moorenstrasse 5, D-40225 Düsseldorf, Germany, and the ¶¶ Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425 

   

 Received for publication, January 14, 2004, and in revised form, April 5, 2004.  

      ABSTRACT       TOP   ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES   RESULTS   DISCUSSION   REFERENCES     To isolate new zinc finger genes expressed at early stages of peripheral nerve development, we have used PCR to amplify conserved zinc finger sequences. RNA from rat embryonic day 12 and 13 sciatic nerves, a stage when nerves contain Schwann cell precursors, was used to identify several genes not previously described in Schwann cells. One of them, zinc finger protein (ZFP)-57, proved to be the homologue of a mouse gene found in F9 teratocarcinoma cells. Its mRNA expression profile within embryonic and adult normal and transected peripheral nerves, and its distribution in the rest of the nervous system is described. High levels of expression are seen in embryonic nerves and spinal cord. These drop rapidly during the first few weeks after birth, a pattern mirrored in other parts of the nervous system. ZFP-57 localizes to the nucleus of Schwann and other cells. The sequence contains an N-terminal Krüppel-associated box (KRAB) domain and ZFP-57 constructs containing green fluorescent protein reveal that the protein colocalizes with heterochromatin protein 1 to centromeric heterochromatin in a characteristic speckled pattern in NIH3T3 cells. The KRAB domain is required for this localization, because constructs lacking it target the protein to the nucleus but not to the centromeric heterochromatin. When fused to a heterologous DNA binding domain, the KRAB domain of ZFP-57 represses transcription, and full-length ZFP-57 represses Schwann cell transcription from myelin basic protein and P 0 promoters in co-transfection assays. Zfp-57 mRNA is up-regulated in Schwann cells in response to leukemia inhibitory factor and fibroblast growth factor 2. 

           INTRODUCTION       TOP   ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES   RESULTS   DISCUSSION   REFERENCES    The major stages in the embryonic development of Schwann cells in rodent peripheral nerves have been defined at the cellular level. Nearly all cells isolated from nerves of embryo day (E) 1 14/E15 rats or E12/E13 mice are Schwann cell precursors ( 1 – 4 ). They are derived from the neural crest and give rise to the immature Schwann cells of late embryonic and early postnatal nerves. These cells are the source of the myelinating or non-myelinating cells of adult nerves. Schwann cell precursors can be distinguished from immature Schwann cells on the one hand and from neural crest cells on the other by a number of criteria, including differences in the regulation of survival and DNA synthesis and antigenic phenotype ( 5 – 8 ). 

   

  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 the formation of the glial lineage ( 11, 12 ). It is also likely to be important in later stages of development, because it has the capacity to synergize with Krox-20 (Egr-2) (see below) to activate the connexin 32 promoter ( 13 – 15 ). Two transcription factors, the POU domain proteins Oct-6 (SCIP, Tst-1, Pou3f1) and Brn-2 are involved in the timing of myelination ( 16 – 18 ), whereas Schwann cells in nerves of mice that are null for the zinc finger protein Krox-20 are arrested at the 1:1 promyelinating stage and fail to develop myelin, implying a crucial role for this transcription factor in the initiation of myelination ( 13 ). Recently, gene array technology has been used to identify some of the genes that are regulated by Krox-20. These include many myelin protein and lipid genes ( 19 ). Krox-20 also has a key role in removing Schwann cells from the cell cycle and in inactivating developmental death signals at the onset of myelination ( 20 ). In an in vitro neuron-Schwann cell co-culture system the transcription factor NF B is also required for myelin formation ( 21 ). 

  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. They include Krox-20 (above), and the related proteins Krox-24 (Egr-1) and Egr-3. Krox-24-null mice do not have any obvious peripheral nerve phenotype although the gene is strongly expressed in embryonic development and in adult life in non-myelinating Schwann cells ( 26 ). 

  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 as the KRAB domain, which acts as a powerful transcriptional repressor even when fused to a heterologous DNA binding domain (DBD) in a variety of systems ( 28 – 30 ). In the case of many KRAB domain zinc finger proteins, this domain is known to associate with a co-repressor protein KAP-1 (TIF1 /KRIP-1) ( 30 – 32 ). KAP-1 in turn associates with heterochromatin protein (HP) 1, a dose-dependent mediator of heterochromatin-mediated gene silencing ( 33 – 36 ). In 3T3 and CHO cells HP1 is normally highly enriched in centromeric heterochomatin although it is present in euchromatic regions as well ( 33 ). The fourth identified zinc finger protein in Schwann cells, Kzf1, is a KRAB domain protein that was identified as a Krox-20 responsive gene in a gene array screen but has not been studied further ( 19 ). 

  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 based on RT-PCR, using degenerate primers recognizing the conserved zinc finger sequence to identify several genes containing zinc finger motifs not previously described in Schwann cells. The properties and distribution of one of these, Zfp-57, is described below. This gene, which contains a KRAB domain, shows strong developmental regulation both in the peripheral and central nervous systems, localizes to heterochromatin in cell nuclei and represses activation of myelin promoter and reporter constructs in co-transfection assays. 

        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 and E13 rat sciatic nerves using TRIzol reagent according to the manufacturer's protocol ( Invitrogen ). The analysis of RNA integrity and cDNA synthesis has been described previously ( 37 – 38 ). Zinc finger motif-containing sequences were amplified by PCR using standard reaction conditions for Pfu DNA polymerase (Promega) including 25 pmol of primer CysX 2 (5'-TGCCCNGAGTGYGGNAAR-3'), H/C (5'-NGGCTTCTCNCCNGTATG-3'), and 1 µl of the first strand cDNA synthesis reaction. The amplification conditions were as follows: 1 initial cycle for 3 min at 94 °C, followed by 40 cycles for 1 min at 94 °C, 1 min at 48 °C and 1 min at 72 °C, and a final extension for 10 min at 72 °C. Control reactions included water or total RNA. PCR reaction products were separated on a 2% agarose gel in 1 x Tris acetate/EDTA, stained with ethidium bromide, and documented by Polaroid photography. Single bands, representing multiples of the zinc finger motif, were isolated using Qiaex (Qiagen, West Sussex, UK) and directly subcloned into the pCR2.1-vector (TA cloning kit, Invitrogen ) to generate a zinc finger motif-enriched cDNA library. Individual clones were randomly selected and sequenced. 

  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 prepared from total RNA of E12 or newborn animals. The radiolabeled probes were generated using a PCR-based amplification method described by Kendall et al. ( 39 ). The hybridization was carried out at 60 °Cin0.5 M sodium phosphate, pH 7.2, 7% SDS, 2 m M EDTA. Posthybridization filters were washed twice in 2 x SSC, 0.1% SDS at 65 °C and exposed to a Fuji phosphorimager. 

  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/ ) and Phosphobase 2.0 ( www.cbs.dtu.dk ). 

   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'-GAATGGCATTACCAAACAATGGCAGCTAGG-3') and ZFP57-DSP2 (5'-TCAGTCCGAATCTTCTGTCAC-AGGACT-3') with cDNA derived from 10 ng of total mouse or rat testis RNA. The Zfp-57 amplification products were T nucleotide-tailed during the final extension step with 2 units of Taq DNA polymerase, gel-purified, and subcloned into the plasmid pCR2.1 ( Invitrogen ). Six individual transformants of plasmid pCR2.1-rZFP57 were selected and sequenced on both strands to generate an overlapping sequence. All plasmids harboring rat Zfp-57 showed homology to the originally reported mouse sequence. 

   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 PCR amplification with the primer pairs ZFP57-F/R11 and F11/ZFP57-R. Aliquots of each of the corresponding products were added as template in a final PCR reaction using primers ZFP57-F and ZFP57-R; this latter reaction yielding the full-length mouse Zfp-57 cDNA. Primer sequences were: ZFP57-F 5'-AAGGATCCCTGTCTTAGCTTCCTTTGTGC-3', R11 5'-TTCTAGAAATCTCTGATTGTTCTTTCCTG-3', F11 5'-AAGAACAATCAGAGATTTCTAGAAGTGGG-3', and ZFP57-R 5'-AAGGATCCCTGTCTTAGCTTCCTTTGTGC-3'. Internal primers F11 and R11 were complementary at their 5'-ends, and each of these primers runs across exons 1 and 2. The full-length cDNA was purified using the QIAEX II gel extraction kit (Qiagen), digested with BamHI and EcoRI, and cloned into pGEM®-3-Zf(+) (Promega). The construct was verified by sequencing. 

   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 analysis were selected for amplification of mRNAs from both mouse and rat. The sequence for rat Zfp-57 was obtained from our sequencing results. The sequences of mouse Zfp-57 (gi:431181), mouse Krox-20 (gi:16758439), rat Krox-20 (gi:23956051), mouse P 0 (gi:6678927), rat P 0 (gi:8393777), 18 S rRNA (gi:57149), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) (gi:193423) are deposited in the National Center for Biotechnology Information data base. Primers were: ZFP57-USP (5'-CAAGCACCAGAGCACTCACAGGG-3'), ZFP57-DSP (5'-TGGATTCTCTGGTGTATAATGAGG-3'); GAPDH-USP (5'-CCTTCATTGACCTCAACTACATGGT-3'), and GAPDH-DSP (5'-TCATTGTCATACCAGGAAATGAGCT-3'). Primers for Krox-20, P 0, and 18 S RNA were as described previously ( 41, 42 ). Steady-state mRNA expression levels were determined by RT-PCR in 50-µl reactions containing 20 m M Tris-HCl, pH 8.4, 50 m M KCl, 1.5 m M MgCl 2, 0.2 m M dNTPs, 25 pmol each primer, and 2.5 units of Taq DNA polymerase. An initial denaturation cycle of 3 min at 94 °C was followed by 28 x ( Krox-20 ), 30 x ( Zfp-57 ), or 32 x cycles ( P 0 ), of30sat94 °C,30sat52 °C and 1 min at 72 °C before a final extension period of 2 min at 72 °C. Samples containing water or RNA were included as controls. Equal cDNA use was confirmed by amplification of 18 S rRNA by PCR ( 37 ). For the postnatal time course, primers were as follows: ZFP57-USP (5'-GCATTACCAAACAATGGCAGCTAG-3'), ZFP-57-DSP (5'-AGGCCTCTCTCCACGATGCTGAGCC-3') for Zfp-57, and GAPDH-USP (5'-CCTTCATTGACCTCAACTACATGGT-3'), GAPDH-DSP (5'-TCATTGTCATACCAGGAAATGAGCT-3') as an invariant control. 

  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'-ACCACAGTCCATGCCATCAC-3' and GAPDH-R: 5'-TCCACCACCCTGTTGCTGTA-3', amplifying a product of 452 bp, STAT3-F: 5'-TGGACCGTCTGGAAAACTGG-3' and STAT3-R: 5'-ATTTCCGAGACCCTCTGAGG-3', yielding a product of 372 bp, and ZFP57–8F: 5'-TGGGCACCAACAAATTGTGG-3' and ZFP57–7R: 5'-ACTGGTGATTAAAGAGGAAGGT-3', amplifying a 406-bp fragment. 

  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 out at 95 °C for 2 min, followed by 32 cycles at 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s, followed by a final extension at 72 °C for 3 min. 

   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 containing the N-terminal sequence RGSHHHHHH fused to amino acids 5–421 of the ZFP-57 protein. This construct was transformed into E. coli strain Top 10F' ( Invitrogen ). Protein induction and purification of fusion protein were performed according to the manufacturer's instructions (Qiagen). The production of fusion protein was monitored by polyacrylamide gel electrophoresis followed by Coomassie Blue protein staining or Western blot analyses using a monoclonal antibody directed against the N-terminal sequence RGSHHHHHH of the fusion protein product ( 43 ). The fusion protein was purified to homogeneity using Ni-NTA columns (Qiagen) and used for commercial antibody production (Froxfield Farms UK Ltd., Hampshire, UK). For further purification of the antibody, full-length ZFP57 was cloned in-frame into pGEX6P2, and expressed in BL21 at 30 °C after induction with IPTG. The fusion protein was purified on a GST-TRAP column using the AKTA system and the antibody purified by affinity chromatography after coupling the purified fusion protein to a N -hydroxysuccinimide-activated HP column according to manufacturer's instructions (Amersham Biosciences). The specificity and tissue distribution of the antibody was tested by Western blot analysis as previously described ( 38 ). 

   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 experiments. 

   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 HindIII restriction site into the protein coding region. This placed Zfp-57 in-frame with the pRc/CMV-His 6 -tagged plasmid and pQE32 (see below). We constructed a similar vector containing Zfp-57 in the antisense orientation. 

  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 ZFP-57 forms were ZFP-57 142–190, where zinc fingers 2 and 3 are deleted, ZFP-57 136–286, where zinc fingers 2, 3, and 4 are missing, and ZFP-57 1–86, where the N terminus of the protein is absent ( Fig. 2 C ). The fragments were digested with BamHI and EcoRI and inserted in-frame upstream of the GFP gene in the pEGFP-N3 vector (BD Clontech UK, Basingstoke, UK). All constructs were verified by sequencing. 

   

          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 a reference, the mouse sequence published in 1994 ( 61 ) is given at the bottom. Sequence differences are shown in bold. The KRAB A domain is shaded in gray, and a possible KRAB B box is shown in italics. The zinc fingers are underlined. B, alignment of the ZFP-57 KRAB domain with KRAB domains from other proteins. Both the KRAB A and divergent B boxes are indicated. C, structure of the ZFP-57 protein with zinc finger domains, the KRAB-DNA binding motif, and the nuclear localization signal ( NLS ) indicated. D, the phylogenetic relationship of ZFP-57 was determined using PBLAST ( 40 ). Protein sequences were retrieved from the SwissProt data base ( www.ebi.ac.uk ). Sequences are ZFP-57 (gi:431181), Krox-20 (gi:2829416), Krox-24 (gi:119243), ZFP-70 (gi:20141062), ZFP-177 (gi:2501719), ZFP-189 (gi:2501719), ZFP-433 (gi:30173446), ZFP-436 (gi:20141030), ZFP-647 (gi:20141866), KOX6 (gi:28202263), HZF22 (gi:1731419), MZF-1 (gi:12644185), FPM-315 (gi:24418868), KIAAA1559 (gi:20140808), XLCGF48.2 (gi:141649), XLCOF7.1 (gi:141719). They were aligned using ClustalW, and the rooted phylogenetic tree was calculated using the same program.

       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 amplifying the sequences upstream and downstream of the corresponding deletion with primer pairs F1/R2 and F2/R1 in the case of Zfp-57 142–190 and F1/R3 and F3/R1 in the case of Zfp-57 136–286. In each case, primers R2/F2 and R3/F3 carried complementary sequences at their 5'-ends. To obtain the final sequences, the two PCR products obtained for each construct were used as templates in a final PCR reaction using F1 and R1 as primers. The primer sequences were: F1 and R1 (see above), R2 5'-CTTGTTTTGAAAGAAAGGCCTCTCTCCAC-3', F2 5'-CCTTTCTTTCAAAACAAGCCAGCAGCTAGC-3', R3 5'-GTCTGAAACGATGCTGAGCCTTGTAGCC-3' and F3 5'-GCATCGTTTCAGACGTCAACCCAACCAGC-3'. The Zfp-57 1–86 construct was obtained using the PCR primers F4 5'-GGAATTCGCCACCATGGGACCATTCTTTTTCTGTCTGACC-3' and R1. 

  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 (amino acids 1–147) in pBXG-1, as a BamHI/XbaI fragment. Each fragment was obtained by PCR using Pfu polymerase and the primers F5 5'-AAGGATCCGCAGCTAGGAAACAGTCTTCCCAGC-3' and R5 5'-GATCTAGAGAAAAAGAATGGTCCACCTTTGAATACC-3'; or F6 5'-AAGGATCCTGTCTGACCTGTGGCAAATGTTTC-3' and R6 5'-GATCTAGATCAGTCCGAATCTTCTTCTGTCAC-3' and verified by sequencing. Other plasmids used were pG4MpolyII, containing amino acids 1–148 of the GAL4 DBD, GAL4-ERE-tk-CAT, a chloramphenicol acetyltransferase (CAT) reporter construct containing two GAL4 binding sites and an estrogen receptor (ER) element in front of a thymidine kinase promoter-CAT fusion, and VP16-ER(C), a chimeric activator with the DBD of ER and the activation region of VP16 (amino acids 411–490). These constructs have already been described ( 46 ). pG4MpolyII-hsKOX1 (amino acids 1–119) was used as a control for repression ( 30, 47 ). The plasmid pC-H110 (Amersham Biosciences) carrying the lacZ gene was used in all the transfections. To confirm protein expression of the GAL4 fusion constructs, Western blotting was performed as described previously ( 20 ), using an antibody recognizing the GAL4 DNA binding domain (clone 2GV-3, Euromedex.com, Mundolsheim, France). 

  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 analysis (see below). 2 The 0.5-kb fragment of the phosphoglycerate kinase ( PGK ) promoter was derived from a mouse PGK genomic clone C128, a gift from Dr. Antonello Mallamaci. The clone was amplified by PCR from mouse genomic DNA, and the sequence was confirmed with the data base. A SmaI/XhoI fragment ( 500 bp) containing the proximal PGK promoter was ligated upstream of the luciferase reporter gene in the plasmid pXP1. 

   

   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), 100 units/ml penicillin, and 100 µg/ml streptomycin ( Invitrogen ) in 5% CO 2, 95% air at 37 °C. Conditions for transient transfections were essentially as described ( 38 ). BrdUrd was used to measure proliferation in triplicate determinations ( 2 ). 

  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 cultures at the time points indicated. 

  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 were subsequently transfected with 0.5 µg of each DNA construct using LipofectAMINE ( Invitrogen ) following the manufacturer's instructions. Twenty-four hours after transfection, the cells were fixed in 4% paraformaldehyde/phosphate-buffered saline for 10 min and stained with Hoechst dye to visualize heterochromatin within the nucleus ( 36 ) for 5 min before visualization under a Leica DMRE fluorescence microscope fitted with an SP2 confocal head and Leica confocal software. An HCX PL APO x 63, 1.32 NA oil immersion lens was used. For co-localization experiments with HP1, cells containing ZFP57-GFP constructs were fixed as above then labeled with monoclonal antibody to HP1 (1:400) overnight ( 49 ), followed by goat anti-mouse Ig rhodamine (1:100) (MP Biomedicals, Inc) for 30 min, before examination in the confocal microscope. For some co-localization experiments, neonatal mouse Schwann cells were labeled sequentially with antibody to ZFP-57 (overnight), goat anti-rabbit Ig fluorescein, antibody HP1 (overnight), and goat anti-mouse Ig rhodamine. 

  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, and -galactosidase and CAT assays were conducted as described previously ( 50 ). These transfection experiments were carried out three times. 

  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 70–80% pure. 

  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 antibody complement-mediated cell lysis, as previously described ( 1, 48, 52 ). For response to LIF, Schwann cells were immunopanned after serum purification and cultured in DMEM containing 0.5% FCS and 10 ng/ml -neuregulin-1 (Merck Biosciences) for 24 h. The medium was replaced 2 h prior to addition of LIF (20 ng/ml) for various time periods. To test for inhibition of the Janus-activated kinase (JAK)/stress-activated kinase (STAT) pathway AG490 inhibitor (Merck Biosciences) (25 µ M ) was added to some cultures 20 min prior to addition of LIF. 

  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 pituitaries ( 53, 54 ). The day before transfection 2.5 x 10 5 cells were plated in 6-cm poly- L -lysine-coated dishes (Sigma). Schwann cells were transfected using Ca 3 PO 4 precipitation as described ( 55 ). Normally 12 µg of DNA was transfected per dish, consisting of 2.5 µg of supercoiled reporter plasmid, 2 µg of expression vector, 5 µg of supercoiled pBluescript SK (+) DNA as a carrier, and 2.5 µg of a pRSVgal plasmid as a measure of transfection efficiency. The amount of supercoiled reporter plasmid transfected was usually normalized for the molar ratio. Cells were harvested 60 h after transfection and then assayed for luciferase, -galactosidase, and protein (Bio-Rad) as previously described ( 48, 56 ). All transfections were performed in triplicate and, in most cases, were repeated several times with separate plasmid preparations (Qiagen). The relative luciferase activity was expressed as the mean ± S.E. for n repetitions. 

   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 equilibrated for 20 min in phosphate-buffered saline, 0.1% Triton X-100. They were then blocked for 1 h at 4 °C in 0.5% blocking solution (Roche Diagnostics, Lewes, UK), 10% sheep serum, 0.1% Triton X-100. ZFP-57 antibody (1:400) was diluted in 0.2% blocking solution, 10% sheep serum, and 0.1% Triton X-100 were applied overnight at 4 °C. After washing in PBS, donkey anti-rabbit Ig Cy3 or Cy2 (1:200) (Jackson ImmunoResearch Labs Inc, West Grove, PA) was applied for 30 min prior to washing and staining with DAPI solution (0.002% in saline) for 5 min. Preparations were viewed in a Zeiss fluorescent microscope. Cultured Schwann cells were plated on to laminin-coated coverslips ( 58 ). After a 5-min fixation in 4% parafor-maldehyde, cells were double immunolabeled with rabbit antibodies to ZFP-57 and mouse monoclonal antibody 192 IgG to the p75 neurotrophin receptor (p75NTR) ( 59 ), followed by donkey anti-rabbit Ig Cy2 and goat anti-mouse Ig Texas Red (1:100) (Organon Teknika NV, Turnhout, Belgium), using a similar protocol. 

        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 containing zinc finger motifs, the zinc finger consensus sequence (Tyr/Phe)- X -Cys- X m - Cys- X 3 -Phe- X 5 -Leu- X 2 -His- X o -His- X n ( m is2or4; n is often 5; o is3or4; X is a variable amino acid) was used to design a degenerate primer pair. The primer pair was used to amplify expressed genes containing a zinc finger motif from embryonic rat sciatic nerves. Typically, a ladder of PCR products was generated in which individual bands represented sequences containing different numbers of zinc finger units, because of the modular structure of zinc finger genes built by single or tandem arrays of the C 2 H 2 motif ( e.g. Ref. 60 ) ( Fig. 1 A ). RT-PCR analyses using cDNA derived from the developing mouse sciatic nerve at E12 (early Schwann cell precursor stage) and E13 (late precursor stage) ( 1, 4 ) resulted in a reproducible pattern of amplification products. Within the resolution power of conventional agarose gel electrophoresis, up to 6 repeats of the C 2 H 2 motif were usually observed ( Fig. 1 B ). This result demonstrated the existence of expressed sequences with up to 6 zinc finger motifs in the embryonic sciatic nerve. No amplification products were generated from untranscribed RNA controls indicating that genomic sequences were not amplified by this procedure. The identity of individual zinc finger motifs was confirmed by isolating and subcloning bands representing 4–6 zinc finger motifs. Sequencing of 12 randomly selected plasmids derived from randomly picked bacterial colonies revealed in every case sequences containing 4–6 zinc fingers. Furthermore, no product was detected twice, which suggested that the complexity of zinc finger motif-containing genes is high at the time points selected. Zinc finger-containing genes that show a differential regulation in the Schwann cell lineage were identified by a differential screening approach. First, a zinc finger transcription factor motif-enriched library was created by subcloning bands representing 4–6 zinc finger motifs into the pCRII vector. Subsequent screening of more than 100 colonies by a differential colony hybridization, using cDNA derived from E12 nerves and nerves from newborn animals (data not shown), led to the identification of eight zinc finger motif-containing cDNAs that showed differential hybridization signals between E12 and birth. 

   

          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 hypothetical protein can be amplified using primers directed against the consensus sequence (Tyr/Phe)- X -Cys- X m -Cys- X 3 -Phe- X 5 -Leu- X 2 -His- X o -His- X n w here X is any amino acid, m = 2or4, n = 5, and o = 3 or 4. This amplification strategy generates a repetitive pattern reflecting the constant spacing between individual zinc finger motifs. B, typical RT-PCR pattern of sequences containing expressed zinc finger motifs from E12 and E13 total RNA from rat sciatic nerve after reverse transcription. Controls were E12 and E13 total RNA prior to reverse transcription (- RT ) and a no template control.

       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 mouse genomic DNA containing all the exons and introns of Zfp-57. This allowed us to establish the exon-intron junctions (data not shown) as well as to identify three nucleotide changes with respect to the published Zfp-57 sequence ( 61 ). These were found at positions 351–352, where two C bases were observed, as opposed to two A bases, as previously reported ( 61 ), and at position 590, where A was present, compared with the reported G. This latter results in a change in the amino acid sequence from lysine to glutamic acid ( Fig. 2 A ). Attempts to obtain the full-length mouse Zfp-57 coding sequence failed when using flanking primers to amplify the whole region. Different bacterial strains were used for this cloning, with the same result. The full-length product was only obtained when primers spanning through the exon 1–2 junction were used as internal primers. Other internal primers, which did not cover this region, also failed to work. We also cloned the rat protein coding sequence and identified, in addition to the G to A change already identified when sequencing the mouse genomic DNA, a set of other nucleotide changes, resulting in a series of corresponding changes at the amino acid level; these are indicated in Fig. 2 A. ZFP-57 contains five putative zinc finger motifs. Sequence analysis also indicated the presence of a KRAB domain at the N terminus of the ZFP-57 protein ( Fig. 2 A ). A KRAB A box runs from amino acid 15 to 54 encoded by exon 3 ( Fig. 2, B and C ). In many proteins a KRAB A domain is followed by a KRAB B domain. Although the amino acid sequence following the KRAB A domain in ZFP-57 shows relatively few similarities with other KRAB B domains, it is nevertheless possible that it represents a divergent KRAB B domain. This would extend from amino acid 55 to 75, and it would be encoded by exon 4. The C-terminal domain contains a predicted nuclear targeting motif and in F9 cells Zfp-57 localizes to the nucleus ( 61 ). Numerous putative phosphorylation sites for protein kinases (CaMKII, CKI, CKII, PKA, PKC, PKG) were identified (not shown), suggesting that considerable post-translational modification of the protein may occur in vivo. The dendrogram in Fig. 2 D shows that the phylogenetic relationship of ZFP-57 to other zinc finger proteins is only limited. Homologies to reported sequences were below 30% ( Fig. 2 D ). Structural prediction analyses revealed that ZFP-57 is a basic protein of 421 amino acids with a pI of 9.9 and a calculated molecular mass of 47.9 kDa. Interestingly, three amino acids account for almost 30% of the protein (serine 10.2%, arginine 9.5%, lysine 8.1%). 

   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 transcription factor Krox-20 by semiquantitative RT-PCR. Zfp-57 mRNA was clearly expressed at E12, the Schwann cell precursor cell stage. A transient maximum of expression was observed at E17 before the expression fell postnatally to lower levels ( Fig. 3 A ). A similar time course was observed for rat Schwann cell development (data not shown). We examined Zfp-57 expression postnatally in more detail and found that Zfp-57 levels leveled out at 20% of neonatal levels at P28 and subsequently ( Fig. 3, B and C and not shown). In contrast to Zfp-57, Krox-20 mRNA was barely detectable at the Schwann cell precursor stage ( Fig. 3 A ). In confirmation of our previous findings ( 41 ) the expression levels of Krox-20 steadily increased from E17 to P13. 

   

          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 Zfp-57 and Krox-20. Note a transient peak of Zfp-57 expression at E17 and decline thereafter, in contrast to Krox-20. B, Zfp-57 mRNA levels during postnatal rat sciatic nerve development. Total RNA isolated from postnatal rat sciatic nerves at the time points indicated was amplified in a linear range by RT-PCR. Representative RT-PCR signals showing Zfp-57 mRNA steady state levels ( top ) and the coamplified GAPDH RT-PCR signals ( bottom ) used for normalization. C, densitometric quantification of Zfp-57 mRNA levels after normalization against the GAPDH RT-PCR products. At each time point the mean values of four individual PCR experiments (two independent cDNA syntheses) are shown. Error bars represent S.E. D, Western blot analysis of protein lysates from E. coli strain DH5a-induced (+ IPTG ) or non-induced (- IPTG ) to express full-length ZFP-57. Note that the molecular mass of the induced ZFP-57-GST fusion protein is considerably higher (73 kDa) than that of ZFP-57 alone seen in Fig. 4 D. The presence of shorter immunoreactive protein products that arise from preliminary protein translation termination because of the unbalanced amino acid content of ZFP-57 is seen below the upper band. E, immunocytochemical analysis of teased fiber preparations from E16, newborn ( NB ), and P10 mouse sciatic nerve using a polyclonal antibody raised against full-length ZFP-57. Note ZFP-57 immunoreactivity in most of the nuclei visualized by the DAPI nuclear label in E16 and newborn nerves. F, ZFP-57 antibodies bind to nuclei of P3 cultured mouse Schwann cells. Note the punctate pattern of immunolabeling in some cells (see Fig. 5 ). The cells are double immunolabeled with antibodies to p75NTR to identify Schwann cells.

       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-histochemistry the antiserum revealed clear nuclear labeling in most or all Schwann cells at E16 and at birth in teased fiber preparations from sciatic nerve ( Fig. 3 E ). ZFP-57 immunoreactivity was also readily detected in the nuclei of Schwann cells that had been dissociated from newborn nerves and plated in vitro ( Fig. 3 F ). 

  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 loss of differentiation ( 6 ). Zfp-57 mRNA levels in the distal stump of the transected, adult sciatic nerve, where Schwann cells have lost contact with axons, were compared with those in normal nerve. Time points up to 35 days post-transection were analyzed. Similar experiments were done using nerve crush, which allows for nerve repair and onset of re-myelination toward the end of the experimental period, in this case 28 days. Unlike many other Schwann cell-associated genes ( 7 ), Zfp-57 was not significantly regulated under these conditions, the expression remaining at the relatively low levels seen for Zfp-57 in the adult sciatic nerve (not shown). In contrast, and as expected, levels of mRNA for the myelin protein PMP22 rapidly responded to cut and crush by falling and, in the case of crushed nerves, rising again as the nerves regenerated (not shown). 

   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 postnatal time points in mouse tissues by semi-quantitative RT-PCR ( Fig. 4 ). At E12, Zfp-57 was detectable at substantial levels in spinal cord, dorsal root ganglia (DRG), and sciatic nerve. Lower amounts were found in brain. At E15, expression was high and almost comparable in all neural tissues examined. At birth, Zfp-57 was still expressed at abundant levels in spinal cord and brain, whereas levels in DRG and sciatic nerve were significantly reduced. The lung was the only non-neural tissue examined that expressed Zfp-57 throughout the developmental period. 

   

          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 shown. B, total RNA was isolated from the adult organs indicated. To allow detection of ZFP-57 in adult tissues the number of cycles was increased from 32 cycles in A to 40 cycles. C, total RNA was isolated from primary cultures of the cells indicated. All cells were derived from newborn rats and kept in culture for more than 1 week prior to RNA extraction. 18 S rRNA control experiments corresponding to A, B, and C revealed equal input of cDNA into RT-PCR amplification reactions ( e.g. as shown in B ). A control lane without cDNA is shown in A, B, and C. In C, Zfp-57 expression is strongest in glial cells, in particular oligodendrocytes. D, Western blot of selected adult mouse nervous tissues. Note the presence of ZFP-57 protein in spinal cord and sciatic nerve, but not in other tissues.

       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 cycles was increased from 32 to 40 cycles. Under conditions that allowed detection of Zfp-57 in testis, where expression has previously been demonstrated ( 61 ) some expression was detectable in cerebellum. No amplification products were obtained from cortex, midbrain, medulla oblongata, spinal cord, eye, or muscle, a tissue previously shown not to express Zfp-57 ( 61 ). 

  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 postnatal rat tissue. Expression of Zfp-57 was obvious in oligodendrocytes and detectable astrocytes. No expression was seen in neurons, endothelial cells or pericytes. Taken together these results show that, in addition to the Schwann cell lineage, the Zfp-57 gene is widely expressed in neural tissues, particularly in the glial cells of the central nervous system. Finally, we examined expression of ZFP-57 protein in several adult nervous tissues by Western blot. The signal was detected in sciatic nerve and at lower levels in spinal cord ( Fig. 4 D ). 

   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 nuclear compartments of the cell. To localize centromeric heterochromatin we used Hoechst DNA-binding dye and antibodies to HP1. Both the dye and HP1 have been shown to localize to centromeric heterochromatin in NIH3T3 cells, and are seen as bright fluorescent clustered patches within the nucleus ( 33, 34 ). To express wild-type ZFP-57 protein, cDNA for the full-length ZFP-57 protein coding sequence was cloned in-frame with enhanced green fluorescent protein (EGFP) at the C-terminal end. In this construct the expression of ZFP-57/EGFP fusion protein is under control of the human cytomegalovirus promoter. In cells transfected with control construct expressing EGFP alone, EGFP was seen throughout the cell. This indicates that expression of EGFP alone is not sufficient to localize the construct either to the nucleus or to heterochromatin patches within the nucleus. In contrast, ZFP-57 wild-type protein was localized within the nuclei of transfected cells, consistent with the proposed role of ZFP-57 as a nuclear transcription factor and in agreement with results obtained for endogenous ZFP-57 visualized with antibodies ( Fig. 5 A ). Notably, in a high proportion of cells (56%) ZFP-57 was not evenly distributed within the nucleus. Colocalization of the protein both with Hoechst dye and the protein HP1, which is known to be highly expressed in heterochromatin-rich areas ( 33 ), revealed that the protein was clustered and highly enriched in these areas ( Fig. 5 A ). Some green fluorescence seen within the cell cytoplasm is probably caused by the synthesis of the protein in the endoplasmic reticulum but it is also possible that the protein can move between the cytoplasm and the nucleus. To identify regions of the protein that are important for the sub-cellular localization, several mutants were generated on the basis of the structural analyses shown in Fig. 2 C. The mutants generated ( 142–190, 136–286, and 1–86) were transiently transfected into NIH3T3 cells. As shown in Fig. 5, ZFP-57 142–190 and ZFP-57 136–286, which result in the deletion of zinc fingers 2 and 3 and 2, 3, and 4, respectively, showed a similar distribution of protein to the wild-type ZFP-57 protein, i.e. the fluorescence was mainly found in heterochromatin-rich areas (in 54% and in 53% of the transfected cells, respectively), indicating that removal of the zinc fingers does not affect targeting to these areas of the nucleus. In contrast, in the mutant 1–86 protein containing all the zinc fingers but lacking the KRAB domain, the protein no longer selectively co-localized with the heterochromatin-rich areas, although the fluorescence remained mainly nuclear ( Fig. 5 A ). This indicates that the KRAB domain is required for targeting to heterochromatin-rich areas within the nucleus in NIH3T3 cells. Although it is possible that the use of transfection could lead to aggregates of overexpressed protein we consider this unlikely in the case of ZFP-57. We do not see patches of ZFP-57 that are HP1 -negative, as would be expected for artificial ZFP-57 aggregates. In addition, even though the expression level of the ZFP57EGFP varies widely among individual transfected cells, there is no correlation between the expression level and the presence of discrete patches of fluorescence that co-localize with HP1 in the 3T3 nuclei. Discrete patches are seen even in cells with very low levels of fluorescence. 

   

          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 mutant proteins and EGFP control protein as indicated. The middle panels show blue Hoechst staining, which highlights A/T-rich repeat sequences in the heterochromatin areas. The right hand panels show a merger between the EGFP and Hoechst images. In the first set of panels note that the EGFP control construct localizes to both nucleus and cytoplasm and fails to co-localize with the Hoechst dye within the nucleus. In the second set of panels note the overlap of wild-type ZFP-57 fluorescence with Hoechst dye in heterochromatin-rich areas. In the third and fourth panels the removal of zinc fingers does not affect this localization. In the fifth set of panels the removal of the KRAB domain ( 1–86) results in a failure to overlap with the Hoechst dye, demonstrating that the KRAB domain is necessary for targeting to heterochromatin-rich areas. In the bottom panels the overlapping fluorescence between wild-type ZFP-57-EGFP and HP1 immunolabeling confirms the localization to centromeric heterochromatin. B, double immunolabeling of mouse Schwann cells with antibodies to ZFP-57 and HP1. The right hand panel shows extensive overlap of expression between the two proteins.

       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 within the cell nuclei ( Fig. 5 B ). It was clear that in Schwann cells HP1 was generally localized in small punctate foci and that the larger distinct patches of HP1 seen in 3T3 cells were not often seen. This presumably reflects different chromatin organization in the two cell types under the conditions used. Notably, the ZFP-57 localization in Schwann cells was similar to that of HP1. Small punctate foci of endogenous ZFP-57 were seen, in contrast to the larger distinct patches seen in transfected 3T3 cells. The co-expression of HP1 and ZFP-57 in nuclei is likely to reflect the dynamic relationship between chromatin organization, HP1 and transcriptional regulatory proteins containing KRAB domains such as ZFP-57. 

   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-mediated activation of a GAL4-ERE-tk-CAT reporter construct, containing two GAL4-binding sites and an estrogen response element (ERE) in front of a tk promoter-CAT fusion. Reporter activity of the GAL4-ERE-tk-CAT construct was increased 18-fold in the presence of the VP16-ER DBD activator ( Fig. 6 ). 

   

          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 expression construct as indicated, 1 µg of GAL4-ERE-tk-CAT DNA, 100 ng of VP16-ER(C), and 1 µg of pCH110 DNA. Values represent the averages of three transfection experiments after normalization to the -galactosidase activity of pCH110. Note that GAL4 DBD alone does not activate the reporter construct and that the 1–91 construct without the KRAB domain produces little repression. B, Western blot of GAL4 and GAL4-ZFP57-KRAB from a KRAB inhibition experiment, showing comparable expression of the two constructs. Left lane, cells infected with GAL4 ( lower arrow ); right lane, cells infected with GAL4-ZFP57-KRAB ( upper arrow ). C, Western blot of GAL4-ZFP57-KRAB and GAL4-ZFP57 1–91 from a KRAB domain deletion experiment showing comparable expression of the two constructs.

       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 (amino acids 1–147) was at the N terminus of the Zfp-57 sequence. This construct (pBXG1-ZFP57) was then transfected into COS-7 cells, together with GAL4-ERE-tk-CAT and VP16-ER(C). The results of these transfections were compared with control transfections carried out with two GAL4 DBD expression vectors (pBXG1 and pG4MpolyII) that had not been fused to Zfp-57. No difference was observed between the effects of either of these two latter constructs on reporter activity (data not shown). As a positive control construct in parallel repression assays we used hsKOX1-KRAB(AB), where the human KOX1-KRAB(AB) has been fused to the GAL4 DBD in pG4MpolyII ( 30, 47 ). We found that pBXG1-ZFP57 strongly repressed the VP16-mediated activation of the GAL4-ERE-tk-CAT reporter, reducing it to 13.4% of the value obtained with either the pG4MpolyII or the pBXG1 control constructs ( p < 0.001) ( Fig. 6 ). In the presence of the VP16 activator, the positive control construct hsKOX1-KRAB(AB) repressed reporter expression down to 3.9% of the value obtained with the pG4MpolyII construct ( p < 0.005) (data not shown). Similar expression levels were observed for the two unfused GAL4 DBD proteins. As a further control, to verify that the KRAB repression was specific we also used a construct containing amino acids 92–421( 1–91) ( i.e. ZFP57 minus the KRAB domain) ( Fig. 6 ). In the presence of this construct reporter expression was 78.4% of the activated control level, indicating that the KRAB domain repression was specific and that other regions of the protein produced little repression. Expression of the GAL4 fusion proteins was confirmed by Western blotting ( Fig. 6 ). 

   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 0 promoter, 9.0 kb of the MBP promoter, and 0.5 kb of the PGK promoter, each of which was linked to the luciferase reporter gene. Controls included co-transfections with Zfp-57 in the antisense orientation, or the empty CMVH6 vector. All co-transfections included pRSVgal to monitor transfection efficiency. ZFP-57 significantly repressed activation of all three promoters relative to the empty vector or to Zfp-57 in the antisense orientation ( Fig. 7 A ). In other experiments, using the 1.1-kb P 0 promoter and the PGK promoter, the repression by ZFP-57 was shown to be dose-dependent, with half-maximal repression evident with 200 ng of ZFP-57 plasmid ( Fig. 7 B ). 

   

          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 cytomegalovirus construct ( CMV ) expression vector alone on the one hand, and one of three target promoters on the other, i.e. MBP, P 0, and PGK. Note inhibition of promoter activity by ZFP-57, measured by luciferase activity. B, the repressor activity of ZFP-57 toward the P 0 and PGK promoter is dose-dependent.

        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 shown). We therefore tested the effects of the growth factor LIF (which acts through the gp130 receptor and the JAK/STAT3 pathway to maintain stem cells in an undifferentiated state ( 63 )) on Zfp-57 expression in Schwann cells. LIF is up-regulated in Schwann cells when they are deprived of axonal contact and acts together with monocyte chemoattractant protein 1 to summon macrophages to the nerve after transection ( 64 ). 

  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 point from a control culture where no LIF had been added and from another culture where the JAK/STAT inhibitor AG490 had been added to the cells 20 min prior to the addition of LIF. 

  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 activates the STAT pathway in Schwann cells, resulting in a transient increase of Zfp-57 expression. 

   

          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 from Schwann cell cultures treated with 20 ng/ml LIF for 30 min, 1 h, 3 h, or 6 h as indicated. The results obtained from control cultures grown in the absence of LIF and for cultures treated with the AG490 JAK/STAT inhibitor prior to addition of LIF for 1 h are also shown, as is the water control in the first lane.

        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 cells. To further explore a possible relationship between proliferation and ZFP-57 expression we tested whether enforced expression of ZFP-57 protein interfered with cell division. Swiss 3T3 cells were stably transfected with constructs that expressed Zfp-57 as a functional gene (sense orientation) or in an antisense orientation. RT-PCR analysis confirmed the expression of these constructs in the transfected cells (not shown). The third pool of cells was transfected with the empty vector that served as an additional control. Cell growth was determined by BrdUrd incorporation in genomic DNA during S-phase in non-synchronized cultures. No difference was seen between any of the three pools of cells. 

  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-57 did not alter DNA synthesis in a statistically significant manner. In another test of the relationship between ZFP-57 and proliferation, RT-PCR was used to examine Zfp-57 mRNA levels in Swiss 3T3 cells before and after stimulation by a mitogen, FGF-2. Examination of Krox-20 was included for comparison, because it is an immediate-early gene that is rapidly but transiently induced by mitogens in these cells. In serum-starved cells prior to mitogen addition, when nearly all the cells are in the G 0 /G 1 phase of the cell cycle, Zfp-57 mRNA was low. Following addition of FGF-2 (12 ng/ml), Zfp-57 mRNA levels rose with a delay relative to Krox-20 mRNA. They were still elevated at 20 h although the levels of Krox-20 had fallen by that time ( Fig. 9 ), indicating a possible relationship between the cell cycle and ZFP-57 expression under these conditions. 

   

          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 after addition of FGF-2 as indicated.

             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 expressed during embryonic development of the peripheral nervous system. The modular structure of the C2H2 zinc finger motif found in tandem orientation in many transcription factors enabled us to use a modification of a PCR-based genetic screen described previously by Pellegrino and Berg ( 65 ). Here, we demonstrate that mRNA from a limited source can be used as efficiently as genomic DNA as a source to create a zinc finger motif cDNA library. Subsequent differential hybridization of the zinc finger enriched library led to the identification of transcription factors differentially expressed between E12 and birth. This approach is fast and reliable and could find wide-spread use in related fields. 

  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 by approximately one-third of known zinc finger proteins ( 68, 69 ). ZFP-57 is likely to have a KRAB(AB) box, where the B box is highly divergent ( 70 ). KRAB A and KRAB B boxes are encoded by exons 3 and 4. We find a single base pair change in the mouse protein coding sequence from the reported sequence. This change is also present in the rat sequence. Rat ZFP-57 is highly homologous to the previously reported mouse ZFP-57 ( 61 ). ZFP-57 has low homology to other zinc finger transcription factors, and significant homologies are restricted to the KRAB A box. 

  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 GAL4 DBD domain, whereas a construct lacking the KRAB domain produces only slight repression. Consistent with this, in transient transfection assays, ZFP-57 also represses the promoter regions of the Schwann cell myelin-associated genes, MBP and P 0, as well as the promoter of the widely expressed PGK gene even at low doses. 

  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 ). Nevertheless, removal of this domain does not affect the targeting of the protein to the nucleus, perhaps because of the nuclear localization signal present at the C-terminal end of ZFP-57. Significantly, removal of zinc fingers 2, 3, and 4 does not affect the localization to heterochromatin-rich areas. 

  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 levels persisting at E15 in these tissues and appearing in brain. Mouse embryonic stem cells also express ZFP-57 and it has been suggested that inactivation of the STAT3 pathway leads to down-regulation of Zfp-57 in embryonic stem cells. 3 This is consistent with our finding that LIF transiently up-regulates Zfp-57 in Schwann cells, an activation that is blocked by an inhibitor of the JAK/STAT pathway. We have shown previously that LIF also transiently up-regulates monocyte chemoattractant protein-1 in Schwann cells ( 64 ). 

   

  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 overlaps with the up-regulation of key regulators of Schwann cell maturation and differentiation such as Oct-6 and Krox-20 ( 10, 41 ). Previously, expression in developing whole mouse embryos was reported to be highest at E13 ( 61 ). 

  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 "Results: Zfp-57 and Proliferation"). In addition, mRNA levels fail to rise in Schwann cells after nerve transection, at a time when they re-enter the cell cycle and divide to populate the bands of Bungner ( 48 ). Taken together these results suggest that ZFP-57 does not play a key role in regulating the cell cycle. Nevertheless addition of the mitogen FGF-2 to Swiss 3T3 cells does result in a rise in Zfp-57 mRNA levels after several hours, suggesting that in this case there may be some cell cycle regulation of the gene. 

  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 (not shown), nor do levels change after nerve transection, indicating that it is not axonally controlled. 

  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 in the nervous system and responds to FGF-2 and LIF in vitro, but is not controlled by axons in peripheral nerves. The predominant expression of Zfp-57 in the embryonic nervous system suggests that the main function of this protein is in early development rather than in postnatal events such as myelination. 

         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 " advertisement " in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.   

   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 acetyltransferase; ER, estrogen receptor; MBP, myelin basic protein; PGK, phosphoglycerate kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FGF-2, fibroblast growth factor 2; P, postnatal day; JAK, Janus-activated kinase; STAT, stress-activated kinase; p75NTR, p75 neurotrophin receptor; DRG, dorsal root ganglia; EGFP, enhanced green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole; RT, reverse transcriptase; tk, thymidine kinase; IPTG, isopropyl-1-thio- - D -galactopyranoside; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BrdUrd, bromodeoxyuridine.   

   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, Gal4-ERE-tk-CAT, VP16-ER(C), and pG4MpolyII-hsKOX1 constructs, Dr. Antonello Mallamaci (DIBIT, Milan) for the PGK construct, Anna Droggiti for help with CAT assays, and Dr. David Parkinson for discussion, Daniel Ciantar for help with the confocal microscope, Dr. Kate Nobes/Sarah Dickinson for COS-7 cells, and Debbie Bartram for editorial assistance. 

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