J Biol Chem, Vol. 275, Issue 17, 12857-12867, April 28, 2000

Identification of a Novel SCAN Box-related Protein That Interacts with MZF1B
THE LEUCINE-RICH SCAN BOX MEDIATES HETERO- AND HOMOPROTEIN ASSOCIATIONS^*

Tara L. Sander, Amy L. Haas, Michael J. Peterson, and Jennifer F. Morris

From the Kelly Weil Laboratory of Pediatric Molecular Oncology, Medical College of Wisconsin, Departments of Pediatrics and Biochemistry, Milwaukee, Wisconsin 53226

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The SCAN box or leucine-rich (LeR) domain is a conserved motif found within a subfamily of C₂H₂ zinc finger proteins. The function of a SCAN box is unknown, but it is predicted to form -helices that may be involved in protein-protein interactions. Myeloid zinc finger gene-1B (MZF1B) is an alternatively spliced human cDNA isoform of the zinc finger transcription factor, MZF1. MZF1 and MZF1B contain 13 C₂H₂ zinc finger motifs, but only MZF1B contains an amino-terminal SCAN box. A bone marrow cDNA library was screened for proteins interacting with the MZF1B SCAN box domain and RAZ1 (SCAN-related protein associated with MZF1B) was identified. RAZ1 is a novel cDNA that encodes a SCAN-related domain and arginine-rich region but no zinc finger motifs. Co-immunoprecipitation assays demonstrate that the SCAN box domain of MZF1B is necessary for association with RAZ1. By yeast two-hybrid analysis, the carboxyl terminus of RAZ1 is sufficient for interaction with the MZF1B SCAN box. Furthermore, MZF1B and RAZ1 each self-associate in vitro via a SCAN box-dependent mechanism. These data provide evidence that the SCAN box is a protein interaction domain that mediates both hetero- and homoprotein associations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Zinc finger genes encode an abundant class of DNA- and RNA-binding proteins that represent an estimated 5% of the genes in the human genome. Many C₂H₂ zinc finger genes have been demonstrated to function as transcriptional regulators and frequently, zinc finger genes are targeted for disruption in a variety of human diseases and cancers. The Krüppel-like subclass of mammalian C₂H₂ zinc finger proteins, first identified in the zinc finger transcription factor TFIIIA, share a conserved link between the last histidine of the preceding finger motif with the first cysteine of the next finger (H-C link) (1). Krüppel-like proteins often contain conserved modular domains outside of their zinc finger motifs. These identified domains include the KRAB (Krüppel-associated box) domains A and B, FAX (finger-associated box) domain of Xenopus, BTB/POZ (broad complex, tramtrack, and bric-a-brac/poxvirus and zinc finger) or ZiN (zinc finger N-terminal) domain, and the SCAN box or leucine-rich domain.

To date, the functions of the KRAB and BTB/POZ domains have been best characterized. The KRAB domain is a conserved stretch of 75 amino acids found in an estimated one-third of Krüppel-like zinc finger proteins (2). The KRAB domain, further subdivided into domains A and B, functions as a potent transcriptional repressor (3-5) and is predicted to fold into two amphipathic helices (2). The KRAB domain from KOX1 interacts with human TIF1 (also named KAP-1, KRAB-associated protein-1) (6, 7) and appears to exert its transcriptional repression activity through this interaction (6, 7). In addition, the KRAB-A domain of Kid-1 interacts with KRIP-1 (KRAB-A interacting protein), which is likely to be the murine homologue of TIF1 and KAP-1 (8). The POZ domain defines a conserved region of approximately 120 amino acids and is found in 5-10% of zinc finger proteins. The POZ domain is a protein interaction motif (9) that mediates both homo- (10, 11) and heterodimerization (12). Several POZ proteins are transcriptional repressors, including the oncoproteins PLZF (13) and BCL-6 (14), and the POZ domain has been shown to function as an autonomous transcriptional inhibitory domain (15). The POZ domain also has been demonstrated to interact with the co-repressors N-CoR, SMRT, Sin3, and histone deacetylase (16-19), suggesting that POZ-containing proteins mediate transcriptional repression by recruiting histone deacetylase through a co-repressor complex. However, this may not be a general mechanism for POZ-containing transcription factors (20).

The leucine-rich (LeR) domain is a conserved motif present in the amino terminus of a subfamily of C₂H₂ zinc finger proteins (4). This motif has also been designated the SCAN box, which was derived from the first four proteins found to contain this domain (SRE-ZBP, CT-fin-51, AW-1, number 18 cDNA) (21). To date, the SCAN box has been identified in approximately 20 zinc finger proteins from human, mouse, and rat including ZNF174 (21), RLZF-Y (22), FPM315 (23), ZNF213 (24), MZF1B¹ and its murine homologue, MZF-2 (25). SCAN box domains are about 80 residues in length, and approximately two-thirds of the amino acids are highly conserved with 80-100% sequence identity. The function of the SCAN box is unknown. Based on protein sequence analysis, the SCAN box is predicted to form two or three amphipathic helices that may be involved in protein-protein interactions (4, 21). However, no proteins have been identified that interact with the SCAN box. Fusion of the SCAN box to a GAL4 DNA binding domain has demonstrated that the SCAN box cannot confer transactivation or repression function onto a heterologous DNA binding domain (4, 21), suggesting that the SCAN box is not an independent transcriptional regulatory domain.

To gain a better understanding of the SCAN box function, we directed our attention to the SCAN box-containing zinc finger protein, MZF1B.^1,2 MZF1B is an alternatively spliced isoform of the zinc finger transcription factor, MZF1 (26) and the human homologue of murine MZF-2 (25). MZF1 is a 485-amino acid protein that contains 13 C₂H₂ zinc finger motifs arranged in a bipartite DNA binding domain. The consensus DNA binding sites have been identified (27), and MZF may regulate the expression of specific genes in a tissue-specific manner (28, 29). MZF1 expression is both necessary for hematopoietic cell differentiation (30) and critical to the regulation of cell proliferation and apoptosis (31-33). MZF1B cDNA encodes a 734-amino acid protein that shares identity to the carboxyl terminus of MZF1, including the 13 C₂H₂ zinc finger motifs. However, MZF1B encodes an additional 257 residues at its amino terminus, which contains a SCAN box domain. Therefore, the amino-terminal domains unique to each isoform may define the distinct functions of MZF1 and MZF1B proteins. We hypothesized that the MZF1B SCAN box is a protein interaction domain. A human bone marrow cDNA library was screened for proteins interacting with the MZF1B SCAN box domain, and RAZ1 (SCAN-related protein associated with MZF1B) was identified.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

pcDNA Expression Plasmids-- MZF and RAZ1 cDNAs were subcloned into the mammalian expression vector, pcDNA3.1 ()/Myc-His (Invitrogen; Carlsbad, CA) using oligonucleotides synthesized by Life Technologies, Inc. (Table I). Plasmid sequences were confirmed by automated sequencing using AmpliTaq DNA Polymerase FS (Applied Biosystems Inc., Foster City, CA).

Full-length MZF1B cDNA (amino acids 1-734) and the unique amino terminus of MZF1B (amino acids 1-257) were amplified by polymerase chain reaction (PCR)³ methods with oligonucleotides 1 and 2 or oligonucleotides 1 and 3, respectively, and subcloned into the EcoRI and HindIII sites of pcDNA3.1 (A) to produce plasmids MZF1B-mh/pcDNA and MZF1B N-term-mh/pcDNA, where "mh" indicates fusion of the protein to the carboxyl-terminal Myc-His epitope tag.

MZF1B lacking the Myc/His tag was created by subcloning the same PCR fragment from MZF1B-mh/pcDNA into pcDNA3.1 (C) to create MZF1B₃*/pcDNA, which added three extra amino acids (Lys, Ala, Thr) to the carboxyl terminus of MZF1B. To eliminate these extra amino acids, a SmaI fragment from gp-end*/pcDNA (MZF1B amino acids 463-734) was subcloned into the SmaI-digested MZF1B₃*/pcDNA plasmid to create MZF1B*/pcDNA. The plasmid gp-end*/pcDNA was produced by PCR amplification of MZF1B cDNA (amino acids 463-734, including the stop codon) with oligonucleotides 4 and 5 and subcloning the BglII and HindIII PCR fragment into pcDNA3.1 (A).

The unique amino terminus of MZF1B (amino acids 1-257) lacking the Myc/His tag was constructed by subcloning the same PCR fragment of MZF1B N-term-mh/pcDNA into pcDNA3.1 (C) to create MZF1B N-term*/pcDNA, which encodes an extra three amino acids (Lys, Ala, Thr) at the carboxyl-terminal end.

Full-length MZF1B with the SCAN box domain deleted, MZF1B SCAN-mh/pcDNA, was constructed by a three-step subcloning procedure. First, the cDNA encoding amino acids 1-46 of MZF1B was PCR-amplified using oligonucleotides 1 and 6 and subcloned into the EcoRI and KpnI sites of pcDNA3.1 (A), creating MZF1B 1-46-mh/pcDNA. Second, the MZF1B cDNA encoding amino acids 124-357 (contains an internal BamHI site) was amplified by PCR using oligonucleotides 7 and 8 and subcloned into the KpnI and HindIII sites of MZF1B 1-46 mh/pcDNA to create MZF1B 1-357SCAN-mh/pcDNA. Third, a BamHI and HindIII fragment of MZF1B-mh/pcDNA (internal BamHI and 3' HindIII site immediately following amino acid 734) was then subcloned into the BamHI and HindIII sites of MZF1B 1-357SCAN-mh/pcDNA to construct MZF1B SCAN-mh/pcDNA.

The unique MZF1B amino terminus with the SCAN box deleted, MZF1B N-term SCAN-mh/pcDNA, was constructed by subcloning a BglII fragment from MZF1B 1-357SCAN-mh/pcDNA into the BglII-digested MZF1B N-term-mh/pcDNA plasmid.

Full-length MZF1 cDNA (amino acids 1-485) was PCR-amplified with oligonucleotides 9 and 2. The PCR fragment, flanked by BglII and HindIII, was subcloned into the BamHI and HindIII sites of pcDNA3.1 (A) to create MZF1-mh/pcDNA.

The cDNA isolated from yeast two-hybrid screening was removed from the pACT2 expression vector by digestion with BglII (including the hemagglutinin (HA) tag) and subcloned into the BamHI site of pcDNA3.1 (A) to generate ha-RAZ1/pcDNA.

-gal-mh/pcDNA, an expression plasmid encoding Myc-His epitope-tagged -galactosidase, was purchased from Invitrogen (Carlsbad, CA).

Yeast Two-hybrid Expression Plasmids-- MZF1B and RAZ1 cDNA were subcloned into yeast expression cloning vectors, pAS2-1 and pACT2 (CLONTECH; Palo Alto, CA), to generate fusion proteins with the GAL4 DNA binding domain (BD; amino acids 1-147) or GAL4 activation domain (AD; amino acids 768-881), respectively. The constructs were confirmed by automated sequencing using AmpliTaq DNA Polymerase FS (Applied Biosystems Inc., Foster City, CA).

To generate the bait plasmid, SCAN/pAS2-1, the cDNA encoding the MZF1B SCAN box (amino acids 47-123) was amplified by PCR using oligonucleotides 10 and 11 and subcloned into the EcoRI and HindIII sites of the mammalian expression vector CB6+ (34) to create SCAN/CB6+. SCAN/CB6+ was digested with EcoRI and BamHI and the MZF1B SCAN box insert was subcloned into pAS2-1.

To generate fusions of the MZF1B SCAN box with the GAL4 AD and HA tag, the cDNA encoding the MZF1B SCAN box (amino acids 47-123) was amplified by PCR using oligonucleotides 12 and 13 and subcloned into the EcoRI and XhoI sites of pACT2 to create SCAN/pACT2.

RAZ1/pACT2 is the library clone isolated from the yeast two-hybrid screen as described below. This clone contains the RAZ1 cDNA (amino acids 1-217) library insert subcloned into the EcoRI and XhoI sites of pACT2 to generate a fusion protein containing the GAL4 AD and HA tag.

RAZ1 cDNA encoding amino acids 38-217 was amplified by PCR using oligonucleotides 14 and 15 or oligonucleotides 16 and 17 and subcloned into the EcoRI and SalI sites of pAS2-1 or the EcoRI and XhoI sites of pACT2 to create RAZ1 38-217/pAS2-1 and ha-RAZ1 38-217/pACT2, respectively.

cDNA encoding either the amino terminus (amino acids 38-132) or carboxyl terminus (amino acids 133-217) of RAZ1 was PCR-amplified using oligonucleotides 16 and 18 or oligonucleotides 19 and 17 and subcloned into the EcoRI and XhoI sites of pACT2 to generate ha-RAZ1 N-term/pACT2 and ha-RAZ1 C-term/pACT2, respectively.

GAL4 Yeast Two-hybrid Analysis-- The MATCHMAKER Two-Hybrid System 2 and GAL4 Human Bone Marrow MATCHMAKER cDNA library were purchased from CLONTECH. A large scale, sequential transformation of the GAL4-SCAN bait fusion (SCAN/pAS2-1) and bone marrow cDNA library was carried out according to the manufacturer's directions. Briefly, the bone marrow cDNA library (50 µg) was introduced into CG-1945 yeast previously transformed with SCAN/pAS2-1, and yeast transformants were plated onto synthetic drop-out medium depleted of histidine, tryptophan, and leucine (SD His/Trp/Leu) containing 5 mM 3-amino-1,2,3-triazole. Yeast clones positive for HIS3 expression were assayed for -galactosidase activity with the colony-lift filter assay. Yeast plasmid DNA was isolated using glass beads and phenol/chloroform extraction (Method 1 in Ref. 63) and pACT2 library plasmids were rescued via transformation of electrocompetent Escherichia coli KC8 (CLONTECH).

Phage Library Screening-- Approximately 5 × 10⁵ plaque-forming units from a gt11 5'-StretchPlus cDNA library synthesized from the human erythroleukemia cell line, K562 (CLONTECH) were plated and transferred to BA-S nitrocellulose filters (Schleicher & Schuell). The filters were hybridized (35) with a ³²P-labeled RAZ1 cDNA probe (nucleotides 115-396) for 20 h at 65 °C and washed under high-stringency conditions with 0.2× SSC, 0.1% SDS at 60 °C.

Northern Blot Analysis-- Total RNA was isolated from tissue culture cells with TRIzol Reagent (Life Technologies). The RNA was separated by electrophoresis through a 1% agarose-formaldehyde gel and transferred to a 0.2-µm Nytran membrane (Schleicher & Schuell). The membrane was hybridized (35) with a ³²P-labeled RAZ1 cDNA probe (nucleotides 115-654) for 1 h at 68 °C using QuickHyb hybridization solution (Stratagene, La Jolla, CA) and washed under high-stringency conditions with 0.1× SSC, 0.1% SDS at 60 °C.

Rapid Amplification of cDNA Ends (RACE)-- First and second strand cDNA synthesis from human bone marrow poly(A)⁺ RNA (CLONTECH), adapter ligation, and 5' and 3' Marathon RACE reactions were performed using the Marathon cDNA Amplification Kit (CLONTECH) according to the manufacturer's directions. Gene-specific primers were synthesized by Life Technologies for 3' RACE (RAZ1 GSP-2 (nucleotides 361-385), 5'-GTA GCG CCT CAG GCC GAA GCT GAA G-3') and 5' RACE (RAZ1 GSP-1b (nucleotides 723-749), 5'-AAC ACG GGG TGG GGT CCC AGG CTC AGG-3').

In Vitro Transcription and Translation (IVT)-- The TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) was used to synthesize radiolabeled proteins for co-immunoprecipitation experiments. In vitro transcription and translation reactions were carried out in a final volume of 25 µl according to the manufacturer's directions using 1 µg of plasmid DNA and 10-20 µCi of [³⁵S]cysteine or methionine (Amersham Pharmacia Biotech).

Immunoprecipitation-- IVT lysate (5-7 µl) and 1 µg of purified rabbit IgG (-His, -Myc, -post-SCAN, -ZF) or a 1:150 dilution of rabbit -HA antisera were incubated at 4 °C for 2-3 h in 500 µl of immunoprecipitation buffer (150 mM NaCl, 10 mM NaPO₄ pH 7.2, 1% IGEPAL, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml aprotinin). The immune complexes were immunoprecipitated with 10% protein A-Sepharose (Amersham Pharmacia Biotech) for 30 min at 4 °C, washed three times with 1 ml of immunoprecipitation buffer, and resuspended in 15 µl of 1× SDS-Laemmli buffer. The gels were fluorographed with Me₂SO and 2,5-diphenyloxazole and visualized by autoradiography.

Antibodies-- Normal rabbit IgG was used as a negative control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Rabbit polyclonal IgG -His and -Myc are specific for the 6× histidine probe and c-Myc epitope tag, respectively (Santa Cruz Biotechnology). Rabbit polyclonal crude antiserum -HA was raised against the HA tag (Babco, Richmond, CA). Rabbit polyclonal -post-SCAN antiserum was generated using amino acids 124-257 of MZF1B. Rabbit polyclonal -ZF antiserum was generated using zinc fingers 1-4 of MZF1B (amino acids 358-462) (32). IgG fractions were purified from both -post-SCAN and -ZF antisera by protein A chromatography (36).

Data Base Searching-- Computer searches were done using the FASTA, BLAST, and MOTIFS algorithms through the Wisconsin Package software (64) or BLAST version 2.0 through the World Wide Web interface. Nucleotide sequences were compared with entries in the GenBank^TM or expressed sequence tag (EST) data bases, while peptide sequences were searched against the Protein Information Resources (PIR) or Swiss-Prot data base.

Chromosome 20 Sequence-- The Homo sapiens clone RP5-1121G12 from the RPCI5 library maps to chromosome 20q11.1-11.23 and has been assigned the EMBL/GenBank^TM accession number AL109965. These data were produced by the Human Chromosome 20 Mapping and Sequencing Groups at the Sanger Center. Mapping and sequence data can be obtained on the World Wide Web.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The MZF1B SCAN Box Interacts with RAZ1-- A human bone marrow cDNA library was screened by yeast two-hybrid analysis for potential MZF1B SCAN box interacting proteins, and one clone was identified (Table II). To confirm the positive protein interaction, we performed two-hybrid assays in the presence and absence of the MZF1B SCAN box domain. The library plasmid did not autonomously activate reporter gene expression, and a positive protein interaction was only observed when both the MZF1B SCAN box and library plasmid were co-transformed into yeast (Table II). To determine whether the interaction was an artifact of the fusion protein partner, we switched the AD and the DNA BD fusion partners for both the MZF1B SCAN box and interacting clone. The interaction between the MZF1B SCAN box and the isolated library clone was not dependent upon the fusion protein partner (Table II). These control experiments confirm that we identified a cDNA library insert positive for MZF1B SCAN box protein interaction. We have named this protein, RAZ1, a SCAN-related protein associated with MZF1B.

RAZ1 Is a Novel SCAN Box-Related Protein-- The cDNA and amino acid sequences of RAZ1 are shown in Fig. 1A. The sequence is not found in the GenBank^TM data base and appears to be a novel clone. The open reading frame for RAZ1 is defined by fusion with the upstream GAL4 activation domain and encodes 217 amino acids, starting with a glycine and ending with a stop codon at nucleotide position 652. The first methionine is at nucleotide 115 and contains a weak Kozak consensus sequence for translation initiation (37). Thus, it is probable that we have isolated a partial cDNA clone from the GAL4 fusion library that is incomplete at the 5'-end. The predicted sequence of RAZ1 encodes a SCAN-related domain at its carboxyl terminus (amino acids 140-200) but no zinc finger motifs. We refer to the domain as "SCAN-related" because the alignment with SCAN domains in other zinc finger proteins is conserved at the amino terminus and truncated at the carboxyl terminus (Table III). Approximately 20 SCAN box-containing proteins have been reported and/or deposited into the GenBank^TM data base that contain zinc finger motifs and/or KRAB domains (Table III). Thus, the SCAN box appears to be frequently associated with zinc finger motifs and sometimes with KRAB domains. It is possible that RAZ1 is a member of a novel gene family of non-zinc finger SCAN proteins. Immediately following the SCAN-related domain is an arginine-rich region (amino acids 201-217). The RAZ1 open reading frame also contains putative sites for post-translational modification: two casein kinase II phosphorylation sites at amino acid positions Ser⁶⁷ and Ser⁷⁷, two protein kinase C phosphorylation sites at positions Thr¹⁰¹ and Thr¹⁴⁴, one cAMP- and cGMP-dependent protein kinase phosphorylation site at position Thr²¹¹, and two N-myristoylation sites at positions Gly⁵⁰ and Gly⁶² (Fig. 1A).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   RAZ1 cDNA and amino acid sequence. A, the isolated RAZ1 cDNA sequence and open reading frame encoding 217 amino acids are shown. A methionine at nucleotide position 115 (amino acid 38) contains a putative translation start site based on Kozak's consensus sequence for translation initiation, (GCC)GCC(A/G)CCAUGG (37). A solid underline indicates the SCAN-related domain at amino acids 140-200. The arginine-rich region is at amino acids 201-217. Several putative phosphorylation (open circles) and myristoylation sites (open squares) are present within the sequence. A schematic diagram of RAZ1 is illustrated below the sequence (not drawn to scale). The cDNA sequence has been deposited into the GenBankTM data base (accession no. AF207829). B, RAZ1 maps to chromosome 20. RAZ1, the cDNA sequence (1-775 plus poly(A)) isolated from yeast two hybrid screening. Chrom 20, a GenBankTM-deposited human chromosome 20 sequence (accession no. AL109965) at 20q11.1-11.23, is identical to RAZ1 cDNA at nucleotides 1-59 and 60-775 and contains an additional 108 bp between nucleotides 59 and 60 of RAZ1. A dotted arrow indicates an open reading frame (ORF). An asterisk indicates a stop codon. AD, activation domain.

View this table: [in this window] [in a new window]   Table III The RAZ1 SCAN domain lacks the predicted third -helix conserved in other SCAN box proteins     SCAN box   NH2-                          Helix 1             Helix 2                    Helix 3  -COOH     consensus:a     ...E..R..FR.F.Y....GP.EAL..LRELC..WLRPE..TKEQILELLVLEOFL.ILP.E.Q..V....PES.EE.V...E.L     RAZ1:b        LGP*TF*QR**Q*R*QDAA**R**FRO****SRO****DIR*****V*M**O**L*A***EAARARRIRRRTDVRITG

RAZ1 Maps to Chromosome 20-- During the course of our studies, we identified a GenBank^TM-deposited human chromosome 20 sequence at 20q11.1-11.23 with identity to RAZ1 at nucleotides 1-59 and 60-775. This sequence also contains an additional 108-bp insert between nucleotides 59 and 60 of RAZ1. An illustration of the chromosome clone is shown in Fig. 1B. To further examine the RAZ1 gene, 5 × 10⁵ clones from a K562 cDNA library were screened with a RAZ1 cDNA probe. Nine unique clones were isolated, and all are identical to RAZ1, extending from nucleotide 21 and through the poly(A) tail (data not shown). In addition, 3'-RACE analysis identified products identical to the 3'-end of RAZ1 that correspond to the contiguous genomic DNA sequence on chromosome 20 including the stop codon, polyadenylation signal, and poly(A) tail. We obtained six 5'-RACE products identical to RAZ1 at the 5'-end, of which one extends from nucleotide 17. Interestingly, one 5'-RACE product contains the 108-bp insert between nucleotides 59 and 60 of RAZ1 (data not shown). These data provide independent confirmation that two RAZ1 transcripts may exist with divergence at the 5'-end, one that does not contain an additional 108 bp of sequence and one that does.

RAZ1 mRNA Is Expressed in Various Cell Lines-- Northern blot analysis detects a RAZ1 transcript of ~1 kilobase in total RNA isolated from both human hematopoietic and nonhematopoietic cell lines. The highest levels of RNA expression were detected in the cell lines HEL (erythroleukemia) and Caco-2 (colon adenocarcinoma) (Fig. 2). The blot was reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to verify equal loading of RNA (Fig. 2). Comparison of RAZ1 with the EST data base identified 100 ESTs between 300 and 600 bp in length that are identical to RAZ1. The ESTs were isolated from various human tissues including brain, breast, fetal heart, kidney, melanocyte, ovarian tumor, and placenta (data not shown). Six of these EST sequences contain the additional 108-bp insert between nucleotides 59 and 60 of RAZ1. Northern blots and reported ESTs suggest that RAZ1 may be widely expressed.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis identifies a RAZ1 transcript of ~1 kilobase in both hematopoietic and nonhematopoietic human cell lines. Lane 1, HL60, human promyelocyte induced to myeloid differentiation with all-trans-retinoic acid; lane 2, KG-1, human myeloblast; lane 3, K562, human erythroleukemia, myeloblast-like; lane 4, HEL, human erythroleukemia; lane 5, U937, human lymphoma, monocyte-like; lane 6, Namawala, human Epstein-Barr virus-positive Burkitt's lymphoma; lane 7, DG75, human Epstein-Barr virus-negative Burkitt's lymphoma; lane 8, Jurkat E6, human T-lymphoblast; lane 9, 293T, human kidney fibroblast; lane 10, HeLa, human cervical adenocarcinoma; lane 11, Caco-2, human colon adenocarcinoma.

The MZF1B SCAN Box Domain Is Necessary for Interaction with RAZ1-- To confirm that full-length MZF1B associates with RAZ1 and to identify MZF1B domains necessary and sufficient for interaction, MZF and RAZ1 proteins were co-expressed in vitro and co-immunoprecipitated with immunospecific antibodies.

As a first step, we demonstrated the immunospecificity of the nonspecific IgG, -His, -HA, -ZF, and -Myc antibodies by immunoprecipitating in vitro expressed MZF and RAZ1 proteins that contain either the amino-terminal HA or carboxyl-terminal mh epitope tag. The pcDNA expression plasmids used for immunoprecipitations are shown in Fig. 3. The antibodies were immunospecific, and no cross-reactivity was observed (Fig. 4A). Full-length MZF1B migrates as an 80-kDa protein, while the MZF1B NH₂ terminus migrates at approximately 42 kDa (Fig. 5). MZF1B SCAN and MZF1 migrate at approximately 72 and 50 kDa, respectively. RAZ1 migrates as a doublet of approximately 35 kDa. We consistently observe that the upper band of RAZ1 is more efficiently immunoprecipitated with -HA. Therefore, the upper band of RAZ1 might be a result of translation initiation at the methionine upstream of the HA epitope tag, while the lower migrating band may be due to internal translation initiation at methionine 38 of RAZ1, thus producing a protein that lacks the HA epitope tag.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   MZF and RAZ1 pcDNA plasmids. The epitope-tagged and nontagged MZF and RAZ1 proteins are shown schematically (not drawn to scale). The mh epitope is fused in frame to the carboxyl terminus of MZF. The HA tag is fused to the amino terminus of RAZ1. The MZF1B SCAN box (amino acids 47-123), acidic domain (MZF1, amino acids 60-72; MZF1B, amino acids 309-321), and RAZ1 SCAN-related box (amino acids 140-200) are shown.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   The MZF1B SCAN box domain is necessary for RAZ1 association. A, controls for antibody specificity. Lanes 1, 5, 9, and 13, 10% of the starting IVT lysate. B, MZF1B associates with RAZ1. Lanes 1-4, MZF1B-mh and ha-RAZ1 were co-expressed in vitro and co-immunoprecipitated with control IgG, -His, or -HA. C, the MZF1B SCAN box is necessary for RAZ1 association. Lanes 5-8, MZF1B SCAN-mh and ha-RAZ1 were co-expressed in vitro and co-immunoprecipitated with control IgG, -His, or -HA. D, MZF1 does not associate with RAZ1. Lanes 9-13, MZF1-mh and ha-RAZ1 were co-expressed in vitro and co-immunoprecipitated with control IgG, -HA, -ZF, or -Myc.

Protein association was demonstrated by co-expressing both MZF1B and RAZ1 epitope-tagged proteins in vitro and co-immunoprecipitating with the immunospecific antibodies. MZF1B is detected when the lysate is immunoprecipitated with -HA, and RAZ1 is detected with -His (Fig. 4B). This suggests that full-length MZF1B and RAZ1 are being pulled down in the same immunocomplex and are interacting in vitro. In addition, neither RAZ1 nor MZF1B SCAN proteins were detected in the same immunocomplex, suggesting that the MZF1B SCAN box is necessary for heteroassociation with RAZ1 (Fig. 4C). Furthermore, MZF1, which lacks the SCAN box domain, does not interact with RAZ1, verifying that the association with RAZ1 is unique to the SCAN box-containing amino-terminal region of MZF1B (Fig. 4D).

The Carboxyl Terminus of RAZ1 Is Sufficient for MZF1B SCAN Box Interaction and RAZ1 Self-association-- To identify RAZ1 domains sufficient for MZF1B SCAN box association, we performed two-hybrid assays with the MZF1B SCAN bait plasmid and either the amino terminus or carboxyl terminus of RAZ1 that contains the SCAN-related domain. As a control, we demonstrated that the individual constructs did not autonomously activate reporter gene expression (Tables II and IV). Positive protein interactions occurred when the MZF1B SCAN box domain was co-transformed with full-length RAZ1 or the carboxyl terminus of RAZ1 but not with the amino terminus of RAZ1 (Tables II and IV). This demonstrates that the carboxyl terminus of RAZ1 is sufficient for MZF1B SCAN box interaction, suggesting that the SCAN box domains from both proteins are mediating heteroprotein association.

To test whether RAZ1 self-associates via a SCAN-dependent mechanism, we performed two-hybrid assays using RAZ1 fused to the GAL4 DNA BD and RAZ1 fused to the GAL4 AD. The individual plasmids did not autonomously activate reporter gene expression (Tables II and IV). Co-transformation of both RAZ1 fusion proteins resulted in colonies positive for protein interaction, suggesting that RAZ1 self-associates in vitro (Table IV). While amino acids 1-37 do not appear to be necessary for RAZ1 self-association, the carboxyl terminus of RAZ1 is necessary for self-association (Table IV). This demonstrates that the SCAN-related domain of RAZ1 mediates homo- as well as heteroprotein association.

The MZF1B SCAN Box Is Necessary for Self-association-- In demonstrating that the SCAN box mediates heteroassociation between MZF1B and RAZ1 proteins as well as RAZ1 homoassociation, we reasoned that the SCAN box might also mediate MZF1B homoassociation. To first determine if MZF1B could self-associate, we performed co-immunoprecipitation assays with epitope-tagged MZF1B and nontagged MZF1B. The Myc/His epitope tag adds ~2 kDa, and the two proteins are distinguishable by size as well as immunoreactivity with the epitope tag-specific antibodies, -Myc or -His. The MZF1B proteins were co-expressed in vitro and co-immunoprecipitated with control IgG, -post-SCAN, or -Myc. In lysates expressing both forms of MZF1B, we detected nontagged MZF1B when epitope-tagged MZF1B was immunoprecipitated with -Myc, suggesting that MZF1B self-associates in vitro (Fig. 5A). To test for the possibility of nonspecific binding, we repeated the assays by co-expressing both MZF1B and epitope-tagged -galactosidase. MZF1B did not associate with -galactosidase, supporting our observation that MZF1B self-association is not an artifact of our co-immunoprecipitation conditions⁴ (data not shown). In addition, the amino terminus of MZF1B is sufficient for self-association, since both epitope-tagged and nontagged MZF1B NH₂ terminus proteins were detected in the same immunocomplex (Fig. 5B). Finally, the MZF1B SCAN box is necessary for MZF1B self-association because nontagged MZF1B NH₂ terminus did not co-immunoprecipitate with MZF1B NH₂ terminus SCAN (Fig. 5C). It should be noted that we consistently observe higher molecular mass bands of >200 and ~80 kDa in immunoprecipitated lysates expressing MZF1B and MZF1B NH₂ terminus, respectively (Fig. 5, A and B). While the identification of these bands has not been confirmed, they may represent higher order complexes of the 80-kDa MZF1B and 42-kDa MZF1B amino terminus.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   The MZF1B SCAN box is necessary for MZF1B self-association. A, full-length MZF1B self-associates. Lanes 1-8, controls for antibody specificity; lanes 9-12, co-expression and immunoprecipitation of tagged and nontagged MZF1B. The IVT reactions for A and B were scaled down to a final volume of 10 µl, and 4.5 µl of the lysate was used for immunoprecipitations in a final volume of 250 µl. B, the MZF1B NH2 terminus is sufficient for self-association. Lanes 1-6, controls for antibody specificity; lanes 7-9, co-expression and immunoprecipitation of tagged and nontagged MZF1B NH2 terminus. C, the MZF1B SCAN box is necessary for self-association. Lanes 1-4, controls for antibody specificity; lanes 5-8, co-expression and immunoprecipitation of nontagged MZF1B NH2 terminus and tagged MZF1B NH2 terminus SCAN. The dotted arrows indicate higher order protein complexes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RAZ1 Protein Structural Motifs-- We have described the identification of RAZ1, a novel human cDNA clone isolated from a yeast two-hybrid screen based on interaction with the MZF1B SCAN box. The function of RAZ1 in unknown, but the predicted sequence contains conserved motifs that provide insight into RAZ1's potential role in regulating transcription factor function. RAZ1 cDNA contains an open reading frame of 217 amino acids with a carboxyl-terminal region homologous to the SCAN box domain conserved in zinc finger proteins. Interestingly, the RAZ1 SCAN box is truncated and lacks the predicted third -helix present in other SCAN box proteins. Thus, we have designated this as a SCAN-related domain. In contrast to other SCAN box proteins, RAZ1 does not appear to encode zinc finger motifs based on the sequence that we have obtained.

The sequences for approximately 20 SCAN box-containing proteins have been reported and/or deposited into the GenBank^TM data base (Table III). Of these, seven also encode a KRAB A and/or B domain, and 19 contain carboxyl-terminal zinc finger motifs. This suggests that the SCAN box is frequently associated with zinc finger motifs and sometimes with KRAB domains. The remaining SCAN box proteins that do not contain zinc finger motifs include p18, TRFA, PGC-2, and RAZ1. p18 and TRFA are partial clones that do not contain sufficient sequence to determine the presence or absence of zinc finger motifs. PGC-2 (peroxisome proliferator-activated receptor  coactivator-2) is a murine adipogenic cofactor bound by the differentiation domain of the peroxisome proliferator-activated receptor  (38). PGC-2 encodes a 142-amino acid protein with a carboxyl-terminal SCAN-related domain but no zinc finger motifs. The PGC-2 protein shares 76% identity to RAZ1 (49% at the NH₂ terminus; 97% at the COOH terminus), suggesting that PGC-2 may be the murine homologue of RAZ1.

It is possible that RAZ1, PGC-2, and potentially p18 and TRFA represent a novel gene family that contain SCAN box domains but lack zinc fingers. Similarly, the SSX gene family contains KRAB domains without zinc fingers. In addition to the KRAB domain, the SSX gene family encodes a novel transcription repression domain at the carboxyl terminus, SSXRD. Interestingly, this SSXRD domain exerts stronger repression than the KOX1 KRAB domain, and the KRAB-related domain fails to interact with the co-repressor TIF1 (KAP-1) (50-52). Therefore, the protein binding and repression function of the SSX genes that contain KRAB domains and lack zinc fingers appears to be different and distinct from KRAB proteins that contain zinc fingers. Thus, SCAN proteins that lack zinc fingers may contain other conserved domains that modify or define their function.

RAZ1 contains an arginine-rich region of 16 amino acids at the carboxyl terminus, immediately following the SCAN-related domain. Short (10-20-amino acid) arginine-rich sequences have been shown to mediate DNA and RNA binding as well as nuclear localization. The arginine-rich domain found in the amino terminus of the recombination activating gene, RAG-1, exhibits DNA binding activity (53). In addition, the arginine-rich motifs of HIV Rev and Tat proteins, bacteriophage  N, 21 N, and P22 N mediate RNA binding (reviewed in Ref. 54). Specifically, the human immunodeficiency virus (HIV) Rev binds to the Rev response element of HIV-1 as an -helix and facilitates the nuclear export of unspliced HIV pre-mRNAs (reviewed in Ref. 54). There is an increasing amount of evidence that the arginine-rich domains present in HIV Rev, Tat, and human retroviruses T-cell leukemia virus type 1 also function as direct importin -dependent nuclear localization signals (55, 56). Thus, the arginine-rich domain of RAZ1 may mediate DNA-RNA binding and/or function as a nuclear localization signal. Localization to the nucleus would place RAZ1 in the same cellular environment as zinc finger transcription factors, and nucleotide binding activity may allow RAZ1 to compete with other zinc finger SCAN proteins for DNA-RNA binding sites. In addition, several putative phosphorylation and N-myristoylation sites reside within RAZ1, suggesting that the function of RAZ1 may be regulated by post-translational modifications.

RAZ1 Gene Structure and mRNA Expression-- The isolated RAZ1 cDNA clone is 775 bp in length and contains a putative translation initiation start site, stop codon, and polyadenylation signal (5'-AAU GAA AAA-3'). Several ESTs, cDNA library clones, and RACE products share identity to RAZ1, suggesting that we have identified a bona fide transcript. In addition, some of the EST and 5'-RACE sequences contain an additional 108-bp insert between nucleotides 59 and 60 of RAZ1. It remains to be determined if both transcripts are expressed in vivo. Northern blot analysis detects an ~1-kilobase RAZ1 transcript in both hematopoietic and nonhematopoietic cells, and ESTs from various tissues share identity to RAZ1, suggesting that the RAZ1 gene is expressed in a variety of tissues. Based on RACE, cDNA library clones, ESTs, and chromosome 20 sequence, it is likely that we have obtained the complete 3'-end of the RAZ1 transcript and are within a few hundred nucleotides of obtaining the entire 5'-end. We scanned the chromosome 20 sequence and found that the open reading frame upstream of RAZ1 continues for 128 amino acids and contains a methionine with a weak Kozak consensus (Fig. 1B). However, further analysis is needed to confirm the complete 5'-end of the transcript.

Interestingly, the RAZ1 gene is localized to chromosome 20q11.1-11.23. Deletion of the long arm of chromosome 20, most often 20q11.2-13, is associated with myeloid disorders, particularly myeloproliferative disorders, myelodysplastic syndrome, acute lymphocytic leukemia, and acute myelogenous leukemia (57-59). This suggests that the genetic loss on chromosome 20q may provide a proliferative advantage to myeloid cells, possibly through the loss of a tumor suppressor gene. In addition, an increased copy number of DNA sequences from chromosome 20q has been observed in pancreatic cancers (60) and breast carcinomas (61), and trisomy of chromosome 20 is associated with the progression of papillary renal cell carcinomas (62). This indicates that a gain of chromosome 20q may facilitate uncontrolled cellular proliferation, possibly through the aberrant expression of an oncogene.

The RAZ1 chromosome 20q11.1-11.23 location raises the question as to whether loss or gain of RAZ1 contributes to any disorders associated with the locus. The myeloid proliferative disorders associated with loss of chromosome 20q are particularly interesting because MZF1/1B appears to be an important regulator of hematopoietic differentiation and proliferation (30-33). Therefore, RAZ1 or MZF1B may be a tumor suppressor gene, and the interaction between RAZ1 and MZF1B may be necessary to elicit a tumor suppressor function. Thus, a genetic loss of RAZ1 might block tumor suppressor activity, thereby providing a proliferative advantage to hematopoietic cells.

The SCAN Box Function-- Co-immunoprecipitation and yeast two-hybrid analyses demonstrate that MZF1B and RAZ1 associate in vitro via a SCAN box-dependent mechanism. In addition, the SCAN box domains are necessary for MZF1B and RAZ1 self-association. Therefore, we have demonstrated that the SCAN box is a protein interaction domain that mediates both hetero- and homoprotein associations. These findings suggest a novel cascade mediated by unique SCAN box protein complexes. To define cellular cascades regulated by SCAN box protein interactions, it will be necessary to identify in vivo SCAN box oligomers and the unique functions elicited by each complex. The identification of mechanisms regulated by SCAN box protein complexes will significantly impact our understanding of the transcriptional role of SCAN box zinc finger proteins and their associated factors.

The transcriptional activity of the SCAN box-containing zinc finger proteins MZF-2 and ZNF174 has been examined. Full-length murine MZF-2 does not activate reporter gene expression, but a truncated form of MZF-2 markedly enhances transcription (29). The transcriptionally active form of MZF-2 contains the SCAN box domain, but the SCAN box is not necessary for transcriptional activity. ZNF174 is a transcriptional repressor of reporter genes driven by the human tumor growth factor- and platelet-derived growth factor-B promoters (21). The complete amino terminus of ZNF174, including the SCAN box domain, transcriptionally represses reporter gene expression when fused to a heterologous DNA binding domain, but the SCAN box domain is not sufficient for transcriptional repression. The ZNF174 repression domain is probably present within the remaining amino-terminal portion, and the SCAN box may or may not modify this function. Thus, the SCAN box does not appear to function as a transactivation or repression domain. These conclusions are supported by our personal observations⁵ and reports by Pengue et al. (4) and Williams et al. (21), which show that the SCAN box does not confer transactivation or repression function onto a heterologous DNA binding domain. While the SCAN box is not an independent transactivation or repression domain, the SCAN box may function to recruit co-repressors and transactivators necessary for transcriptional regulation.

MZF1B Function-- We identified RAZ1 as a potential in vivo protein interaction partner with the SCAN box-containing zinc finger protein MZF1B, the human isoform of MZF1 previously identified by Peterson and Morris^1,2. MZF1 was initially identified as a zinc finger transcription factor necessary for granulocytic differentiation and critical to the regulation of cell proliferation and apoptosis (30-33). In contrast to previous reports, both MZF1B and MZF1 mRNA transcripts are expressed in numerous tissues.² Therefore, previous reports addressing MZF1 function may have been indirect measurements of MZF1B function. Thus, MZF1B may function as an important regulator of granulocytic differentiation, cell proliferation, and apoptosis. The interaction between MZF1B and RAZ1 might be necessary for mediating MZF1B function, or RAZ1 may modify intrinsic MZF1B function. It is also possible that other SCAN box proteins compete with MZF1B for binding to the same protein, thereby providing a transcriptional regulatory mechanism based on the sequestration of specific factors and availability of protein partners. Furthermore, MZF1B and RAZ1 each self-associate in vitro, suggesting that each protein may participate in the formation of unique complexes with distinct functions. Thus, the transcriptional activity of MZF1B is probably mediated by specific protein-protein interactions with RAZ1, MZF1B, and other SCAN box proteins. Identifying the in vivo MZF1B protein partners and their effect on MZF1B activity will provide insight into the possible mechanisms by which MZF1B functions to transcriptionally regulate cell development.

	ACKNOWLEDGEMENTS

We thank Drs. Ronald Hines, Nancy Dahms, and Ravi Misra for reviewing the manuscript, Dr. Bellur Seetharam for providing helpful suggestions and discussions, and Mike Groeschel for excellent technical assistance (Medical College of Wisconsin).

	Addendum

During the course of review for this manuscript, Williams et al. (65) published their finding that the zinc finger-associated SCAN box is a conserved oligomerization domain.

	FOOTNOTES

^* This work was supported by NCI, National Institutes of Health, Grant CA-69141; American Cancer Society Grant ACS-IRG 170; a grant from the Midwest Athletes against Childhood Cancer Fund; a grant from the Bernard and Miriam Peck Foundation; a grant from the Milton and Lillian Peck Foundation; and a grant from the Zink the Zebra Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF055078 (MZF1B), M58297 (MZF1), and AF207829 (RAZ1).

To whom correspondence should be addressed: The Kelly Weil Laboratory of Pediatric Molecular Oncology, Medical College of Wisconsin, Dept. of Pediatrics, MACC Fund Research Center, Rm. 6008, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-4997; Fax: 414-456-6543; E-mail: jmorris@mcw.edu.

¹ M. J. Peterson and J. F. Morris, submitted for publication.

² M. J. Peterson, and J. F. Morris, manuscript in preparation.

⁴ A. L. Haas and J. F. Morris, unpublished results. MZF1B* and -galactosidase-mh proteins were co-expressed in vitro and co-immunoprecipitated with -Myc antibodies. MZF1B* was not in the same immunocomplex as -galactosidase-mh.

⁵ T. L. Sander, A. L. Haas, M. J. Peterson, and J. F. Morris, unpublished observations. When fused in frame to a GAL4 DNA binding domain, the MZF1B SCAN box does not activate the expression of a luciferase reporter gene under the control of a minimal promoter containing five GAL4 binding sites.

	ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; bp, base pair; EST, expressed sequence tag; AD, activation domain; BD, binding domain; HA, hemagglutinin epitope tag; IVT, in vitro transcription and translation; mh, Myc/His epitope tag; RACE, rapid amplification of cDNA ends; HIV, human immunodeficiency virus.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES