, ,1 ,1 ,2

,3

*Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan

�Division of Cardiology, Niigata University Graduate School of Medical and Dental Science, Niigata, Japan

�Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Saitama Japan

§Department of Molecular Neuropathology, Brain Research Institute, Niigata University, Niigata, Japan

¶Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan

More than 100 mRNAs in the zinc-finger gene family whichpotentially regulate gene expression and cell plasticity duringcell differentiation have been isolated from a human braincDNA library (Becker et al. 1995). Expression-sequence taganalysis also demonstrated that over 3% of all genesexpressed in human testis belong to the zinc-finger genefamily (Affara et al. 1994). In the zinc-finger family, theC2H2 class constitutes the largest family of transcriptionfactors or regulators (Bray et al. 1991). The two Cys residuesand two His residues in each C2H2 zinc-finger bind to a zincion to form a structure capable of binding to target DNA(Leon and Roth 2000). Outside of the zinc-finger domain,C2H2 zinc-finger proteins usually bear a conserved domain atthe amino-terminal end which mediates protein–proteininteraction and is often involved in regulating gene expres-

Received August 17, 2009; revised manuscript received October 29,2009; accepted November 24, 2009.Address correspondence and reprint requests to Hiroyuki Nawa,

Department of Molecular Neurobiology, Brain Research Institute,Niigata University, 1-757 Asahimachi-dori, Niigata 951-8585, Japan.E-mail:hnawa@bri.niigata-u.ac.jp1Both these authors contributed equally to this study.2The present address of Takuji Iwasato is the Department of Develop-mental Genetics, National Institute of Genetics, 1111 Yata, Mishima-city,Shizuoka 411-8540, Japan.3The present address of Toshiro Kumanishi is the Niigata LongevityResearch Institute, 766 Shimoishikawa, Shibata, Niigata 959-2516, Japan.Abbreviations used: AP-5, 2-amino-5-phosphopentanoic acid; E8,

embryonic day 8; ES, embryonic stem; EST, expressed sequence tag;KRAB, Kruppel-associated box; NEO, neomycin-resistant gene; RT, re-verse transcriptase; SCAN, SER-ZBP, CTfin51, AW-1 and Number 18cDNA; SDS, sodium dodecyl sulfate; SSC, sodium chloride–sodiumcitrate.

Abstract

To identify a novel regulatory factor involved in brain devel-

opment or synaptic plasticity, we applied the differential dis-

play PCR method to mRNA samples from NMDA-stimulated

and un-stimulated neocortical cultures. Among 64 cDNA

clones isolated, eight clones were novel genes and one of

them encodes a novel zinc-finger protein, HIT-4, which is 317

amino acid residues (36–38 kDa) in length and contains se-

ven C2H2 zinc-finger motifs. Rat HIT-4 cDNA exhibits strong

homology to human ZNF597 (57% amino acid identity and

72% homology) and identity to rat ZNF597 at the carboxyl

region. Furthermore, genomic alignment of HIT-4 cDNA indi-

cates that the alternative use of distinct promoters and exons

produces HIT-4 and ZNF597 mRNAs. Northern blotting re-

vealed that HIT-4 mRNA (�6 kb) is expressed in various tis-

sues such as the lung, heart, and liver, but enriched in the

brain, while ZNF597 mRNA (�1.5 kb) is found only in the

testis. To evaluate biological roles of HIT-4/ZNF597, targeted

mutagenesis of this gene was performed in mice. Homozy-

gous ()/)) mutation was embryonic lethal, ceasing embryonic

organization before cardiogenesis at embryonic day 7.5.

Heterozygous (+/)) mice were able to survive but showing cell

degeneration and vacuolization of the striatum, cingulate

cortex, and their surrounding white matter. These results re-

veal novel biological and pathological roles of HIT-4 in brain

development and/or maintenance.

Keywords: alternative splicing, degeneration, gene targeting,

seizure, vacuolization, zinc-finger protein.

J. Neurochem. (2010) 112, 1035–1044.

JOURNAL OF NEUROCHEMISTRY | 2010 | 112 | 1035–1044 doi: 10.1111/j.1471-4159.2009.06525.x

� 2010 The AuthorsJournal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1035–1044 1035

sion by selectively controlling association of the transcriptionfactors with each other or with other cellular components. Inthe C2H2 class, these modules include the poxvirus and zinc-finger domain, which is also known as the BTB domain(Broad-Complex, Tramtrack, and Bric-a-brack), the Kruppel-associated box (KRAB), and the newly defined SCAN (SER-ZBP, CTfin51, AW-1, and Number 18 cDNA) domain(Collins et al. 2001; Thomas and Emerson 2009). TheKRAB domain is a highly conserved and hydrophobic regioncomposed of �40 amino acids which, in SP1 and ERG1,regulates activation or attenuation of RNA transcription(Oteiza and Mackenzie 2005). The SCAN domain contains aleucine-rich repeat which forms an alpha-helical structureand enables dimerization (Nam et al. 2004). The transcrip-tion factors MZF1 and ZIC2, which regulate tissue devel-opment, exemplify the SCAN domain-containing zinc-fingerproteins (Perrotti et al. 1995; Inoue et al. 2007).

Human genome analysis revealed that nearly two thousandgenes contain the zinc-finger motif, although the molecularfunction of most of these genes remains to be characterized.Here, we report the cloning and characterization of a novelC2H2 zinc-finger protein, HIT-4, whose distribution andfunction have never been characterized yet. As RNA blottinganalysis revealed its unique expression pattern of adult ratbrain, we performed gene-targeted mutagenesis and exam-ined brain pathology of mutant mice.

Materials and methods

Cell cultureNeocortical neurons were prepared from embryonic rats and

maintained in serum-free medium for 1 week as described previ-

ously (Narisawa-Saito et al. 1999). Briefly, whole neocortices of ratembryos (embryonic day 18; E18) were mechanically dissociated

and plated onto poly-D-lysine-coated dishes at a density of 2–

4 · 103 cells/mL. Dissociated cells were grown for 7 days in

Dulbecco’s modified Eagle’s medium containing 2 mM purified

glutamine, nutrient mixture N2 (100 lg/mL transferrin, 5 lg/mL

bovine insulin, 100 nM putrescine, 30 nM sodium selenite, and

20 nM progesterone), 1 mM HEPES, 100 U/mL penicillin, and

100 lg/mL streptomycin.

PCR for differential display and mRNA identificationNeocortical cultures were stimulated with 100 lM NMDA in Mg2+-

free Hanks buffer for 30 min followed by incubation with complete

medium for 7 h. Alternatively, cultures were treated with 100 lM 2-

amino-5-phosphopentanoic acid (AP-5) for 7.5 h. RNA was then

extracted from neocortical cultures and subjected to differential

display PCR according to published procedures (Liang and Pardee

1992). In brief, total RNA was converted to cDNA using 10 lManchor primer of oligo-dT12N (N = dA, dC, or dG) with reverse

transcriptase (RT; Takara Bio, Otsu-city, Japan). Fragments of the

resultant cDNAs were labeled and amplified in the presence of 32P-

dCTP by PCR using the same anchor primer and a set of random

nanomers (10 lM) with five cycles of 94�C/15 s, 40�C/120 s, and

72�C/20 s followed by 35 cycles of 94�C/15 s, 42�C/120 s, and

72�C/20 s and finally 75�C for 5 min. PCR products were subjected

to polyacrylamide gel electrophoresis followed by autoradiography.

PCR fragments that exhibited difference in signal intensity between

NMDA-stimulated, AP-5-treated, and unstimulated samples were

subjected to automatic DNA sequencing (ABI 7700; Applied

Biosystems, Tokyo, Japan).

Reverse transcriptase-coupled PCR was carried out to confirm the

usage of the alternative exons, exon 1A and exon 1B, in HIT-4 and

ZNF597 mRNAs. Forward primers, 5¢-AGACGCAAGACTCCG-CCACT-3¢ and 5¢-CTGGCAGCGGAGTTGGAGAC-3¢ were de-

signed in the exon 1A (HIT-4) and exon 1B (ZNF597) sequences,

respectively. A reverse primer was designed in the common cDNA

sequence for HIT-4 and ZNF597; 5¢-TCCGGTCTTCCCCTTGTC-AAG-3¢. RT-coupled PCR was performed with a RT-PCR reaction

kit (Platinum Quantitative RT-PCR ThermoScript One-Step System;

Invitrogen, Tokyo, Japan). cDNA fragments for HIT-4 and

ZNF597 were synthesized from total whole brain RNA or total

testis RNA (0.5 lg/tube) as a template and amplified in the

presence of the above primers (10 lM) with 16–24 cycles of

94�C/15 s, 60�C/30 s, and 68�C/60 s. PCR products were sub-

jected to agarose gel electrophoresis in the presence of ethidium

bromide (0.2 lg/mL).

Library screening and vector constructionTo isolate full-length cDNA for HIT-4 mRNA, 400 000 plaques of

lambda Zap library carrying rat hippocampal cDNA (Clontech,

Mountain View, CA, USA) were screened with a labeled probe of

the HIT-4 fragment (263 nt) obtained by differential display PCR.

The fragment was labeled by a random priming method (Boehringer

Ingelheim, Tokyo, Japan) and used as a probe (2 · 106 cpm/mL).

The hybridization was carried out at 42�C for 20 h in buffer

containing 50% formamide, 5x sodium chloride–sodium citrate

buffer (SSC), 250 lg/mL denatured salmon sperm DNA, and 5x

Denhardt’s solution, followed by stringent washing at 60�C with

0.3x SSC. Two clones containing larger cDNA were subjected to

automatic DNA sequencing (ABI 7700; Applied Biosystems). One

contained a 5.8-kb insert encoding a full open reading frame of HIT-

4 (951 bp).

To isolate genomic clones for HIT-4, we screened a 129/SVJ

mouse genomic library in lambda DASH II (Stratagene Cloning

Systems, La Jolla, CA, USA) with the above PCR product as a

probe. Among 4.0 · 105 plaques, four clones were positive for the

probe and one clone contained a 17 kb genomic insert with exon 3

in the middle. A KpnI–NotI fragment (�12 kb) of the clone was

subcloned into a Bluescript SK(+) plasmid vector and used for

targeting vector construction. The BglII–BglII fragment (3.7 kb)

containing exon 3 was replaced with a 3-phosphoglycerate kinase–

neomycin-resistant gene (NEO) cassette by blunt-end ligation.

Diphtheria toxin A fragment, a negative selection marker for

homologous recombination, was inserted into the NotI site betweenthe genomic insert and the Bluescript plasmid vector.

RNA analysisTotal RNA was extracted from adult Sprague–Dawley rats (SLC,

Shizuoka, Japan), adult mouse neocortex, or from primary neuronal

cultures by the Guanidium–Phenol method (Amada et al. 2003).PolyA+ RNA was further purified from total RNA by the Oligotex

Journal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1035–1044� 2010 The Authors

1036 | Y. Tanabe et al.

mRNA purification kit (Takara Bio). RNA samples were

denatured in 50% formamide, 6% formaldehyde, 20 mM 3-(N-morpholino)propanesulfonic acid buffer, pH 7.0, 1 mM EDTA at

65�C for 10 min, separated on a 1.5% formaldehyde-agarose gel,

and visualized with ethidium bromide to confirm equal loading of

each sample. Alternatively, a blotting sheet carrying polyA-

enriched RNAs of various tissues was obtained from Clontech

and subjected to hybridization. Following extensive washing with

water, RNA was then transferred onto a nylon membrane

(Amersham-Pharmacia, Tokyo, Japan) in the presence of 10x

SSC buffer and fixed by exposure to ultraviolet light. A 32P-

labeled cDNA probe for HIT-4/ZNF597 mRNA (a 1.4-kb BglII–BglII fragment) was generated using the Random primed DNA

labeling kit (Boehringer Ingelheim). The probe (2–4 · 106 cpm/

mL) was hybridized to filters for 20 h at 42�C in 50% formamide,

5x SSC, 5x Denhardt’s solution, and 1% sodium dodecyl sulfate

(SDS) followed by washing with 0.3x SSC, 0.1% SDS at 60�Cand film exposure.

In situ hybridizationA 1.4-kb BglII–BglII fragment of the full-length HIT-4 cDNA

(GenBank: AF277900.2) covering the cording region was subcloned

into the BamHI site of pBluescript SK()) vector. Digoxigenin-

labeled sense and antisense cRNA probes were made by in vitrotranscription with T3 and T7 RNA polymerases, respectively

(Roche Diagnostics, Indianapolis, IN, USA). Using brain slices

(15 lm thick) of adult rats, we carried out in situ hybridization as

previously described (Watakabe et al. 2007; Abe et al. 2009).

In vitro translationWe subcloned HIT-4 cDNA carrying the putative open reading

frame into Bluescript SK plasmid and synthesized HIT-4 mRNA

with T3 RNA polymerase (Takara Bio) in the presence of a cap

analog, m7ppG. In vitro transcribed RNA was then translated into

protein by the rabbit reticulocyte lysate system (Amersham-

Pharmacia) in the presence of biotinyl lysine. Reaction mixture

(5 lL) was denatured with sample buffer (20 mM Tris, pH 6.8,

2% SDS, and 0.7 M b-mercaptoethanol), separated by

SDS-polyacrylamide gel electrophoresis and blotted onto a

nitrocellulose membrane (Schleicher and Schull, Dassel, Ger-

many). Immunoblots were incubated with streptavidin-peroxidase

diluted in Tris-buffered saline (1 : 1000). Biotin reactivity was

visualized by enhanced chemiluminescence reaction kit (Amer-

sham-Pharmacia).

Generation of HIT-4 knockout miceThe targeting construct was transfected into embryonic stem (ES)

cells (TT2) by the calcium phosphate method and G418-resistant

clones were isolated. Homologous recombinants were identified by

Southern blotting and long PCR. Genomic DNA of cells (5 lg) wasdigested with BamHI and SpeI, separated in a 0.5% agarose gel and

transferred to a nitrocellulose membrane (Schleicher and Schull).

DNA blots were hybridized with 3¢ (a 0.7-kb HindIII–NotIfragment) and Neo cassette (2.5 kb XhoI–XhoI) probes. In addition

to a wild-type 19 kb fragment, the presence of 16 kb fragment

positive for both 3¢ and Neo probes verified homologous recom-

bination. The proper genomic recombination at the 5¢-flankingregion of the targeting vector was confirmed by long PCR (data not

shown).

Embryonic stem cell clones containing the construct were

injected into C57BL/6 blastocysts and chimaeras were derived

(Iwasato et al. 2007). Chimeric male mice were crossed with

C57BL/6 females to achieve germline transmission. Mouse manip-

ulation was performed at the animal facilities of RIKEN-BSI

according to the Institution’s guideline (Saitama, Japan). Back-

crossing to C57BL/6 (over five generations) and breeding were

performed at the animal facilities of Niigata University Brain

Research Institute. All animal experiments were conducted in

accordance with the guidelines of the National Institutes of Health

Guide for Care and Use of Laboratory Animals.

Brain histologyAdult mice were transcardially perfused with 4% p-formaldehyde

and 0.1% glutaraldehyde in a 0.1 M phosphate-buffered solution,

pH 7.4. Serial sections (4 lm thick) were cut from paraffin-

embedded tissues, stained with hematoxylin and eosin or Kluver–

Barrera stain. Alternatively, brain sections were incubated with

mouse monoclonal antibody against 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase (clone 11-5B,1 : 10 000; Sigma, St Louis, MO,

USA) and subsequently stained using the avidin-biotin complex

method (Vector Laboratories, Burlingame, CA, USA).

Results

Genome alignment and amino acid primary structureWe attempted to identify genes whose expression isregulated by activation of NMDA receptors in neurons.Neocortical neurons were prepared from rat embryos,grown in culture, and challenged with 100 lM NMDA or50 lM AP-5 (an NMDA receptor antagonist). Total RNAextracted from cultures was subjected to differential displaywith 60 sets of anchor oligonucleotide primers. Theamounts of RT-PCR products were compared amongNMDA-treated, AP-5-treated, and untreated samples. Dif-ferential display enabled us to identify 40 RT-PCR productswhose amounts were altered by NMDA treatment (data notshown). Among these, we found that the genes for two RT-PCR products were not reported and expressed selectivelyin the brain. We previously characterized one, ATH55, to bea molecule involved in RNA processing (Amada et al.2003). In this study, we report on the identity and functionof the other, HIT-4.

We screened a rat hippocampal cDNA library(3.6 · 105 pfu) using RT-PCR products as a probe andobtained putative full-length cDNA clones. The total lengthof the identified cDNAs was �5.8 kb, which includes 951 bpof open reading frame, 480 bp of 5¢-non-translated region,and a large 3¢-untranslated region. The size of these cDNAclones is in agreement with that of HIT-4 mRNA detected bynorthern blotting (see below). The nucleotide sequence wasinitially deposited to GenBank in 2000 under accessionnumber AF277900. Following the discovery of a homolo-gous cDNA in rat testis, this gene was termed ZNF597 basedon its protein structure (Barbe et al. 2008). However, there is

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Brain defects of HIT-4/ZNF597-knockout mice | 1037

a significant difference in nucleotide sequence at the 5¢-endof HIT-4 and ZNF597 cDNAs (Fig. 1a). The 5¢-end of HIT-4cDNA (204 bp) exhibits no homology to the 5¢-end ofZNF597 (299 bp), although the 3¢-portions of the clones areidentical. The BLAST search of nucleotide sequences of bothclones in rat whole genome (BUILD 3.4) found correspondingexons in chromosome 10 (Fig. 1b). The 5¢-portions of HIT-4and ZNF597 cDNAs are derived from distinct adjacentexons. Thus, HIT-4 and ZNF597 genes have distinctpromoters, different first exons (exon 1A and exon 1B,respectively), and different polyadenylation sites, but sharethe same exon 2 and the protein-coding region of exon 3.

According to the GenBank database, the initiation ofprotein translation in ZNF597 is assumed at the firstmethionine (ATG) in exon 1B. As HIT-4 mRNA lacks thenucleotide sequence of exon 1B (Fig. 1b), we performed anin vitro translation assay to estimate translation initiation siteof HIT-4. HIT-4 cDNA was transcribed from a T3 promoterof its plasmid vector, Bluescript SK()). HIT-4 mRNA wastranslated into protein in rabbit reticulocyte lysates in thepresence of biotinyl-lysine. Western blotting verified a 36–38 kDa translation product from HIT-4 mRNA (Fig. 2). Astranslation initiation from ATG at position 480 in exon 3

theoretically gives a rise to a 36277 Da product, this codon isthe likely translation initiation site.

Rat HIT-4 protein contains seven copies of the zinc-fingermotif, in which four and three copies are clustered at amino-and carboxyl-sites, respectively, separated by 70 amino acids(Fig. 3a). Although ZNF597 was predicted to carry theKRAB transcription regulatory domain at the amino-terminalend (encoded by exon 1B), HIT-4 lacks this domain and istherefore shorter than ZNF597 approximately by one hun-dred amino acids. There is significant amino acid homologyamong individual zinc-finger sequences of HIT-4 protein(Fig. 3b). All zinc-finger sequences contain the commonprimary structure; Cys-X2-Cys-X3-Phe-X5-Leu-X2-His-X3-His, which is often found in transcription factors of the C2H2-class zinc-finger proteins.

Tissue distribution of HIT-4 mRNATo study tissue distribution of HIT-4 mRNA, we carried outRNA blotting and in situ hybridization using a cDNAfragment for the entire protein cording region of HIT-4 as aprobe. Northern blotting of total RNA detected �10- and 6-kb signals in cultured neocortical neurons, which wereprepared from rat embryos (Fig. 4a). Stimulation of cortical

(a)

(b)

Fig. 1 Nucleotide sequence alignment of

rat HIT-4 and ZNF597 cDNAs. (a) The

nucleotide sequences of HIT-4 cDNA

(capital letters) and ZNF597 cDNA (lower

letters) were matched to each other. Iden-

tical sequences are marked with =. Closed

arrows indicate the position of the exon–

intron junction. The boxed ATG represents

the putative translation initiation site. (b)

Nucleotide sequences of HIT-4 cDNA and

ZNF597 cDNA were schematically aligned

to the rat whole genome sequence (BUILD

3.4). The exons for HIT-4 cDNA are indi-

cated with closed boxes and those for

ZNF597 cDNA with open boxes. Putative

translation initiation sites are marked with

open arrowheads.

Journal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1035–1044� 2010 The Authors

1038 | Y. Tanabe et al.

neurons with NMDA enhanced the expression of HIT-4mRNA while an NMDA receptor blocker, AP-5, had noapparent effect. When we examined distributions of HIT-4/ZNF597 mRNAs in adult rat tissues, there were 6- and 1.5-kb signals but not a 10-kb signal (Fig. 4b). The 6-kb signalwas seen in all neural and non-neural tissues with the highestintensity in the brain. The 1.5-kb signal was detected only in

Fig. 2 In vitro translation of transcripts from HIT-4 cDNA. HIT-4 RNA

was synthesized from Bluescript SK plasmid carrying HIT-4 cDNA with

T3 RNA polymerase, then translated in the rabbit reticulocyte lysate

system in the presence of biotinyl lysine. Reaction mixture was sep-

arated by SDS–polyacrylamide gel electrophoresis and blotted onto a

nitrocellulose membrane. Immunoblots were probed with streptavidin-

peroxidase, followed by chemiluminescence reaction. Control experi-

ment was done in the absence of exogenous RNA. An arrow indicates

translation product from HIT-4 RNA. The sizes of molecular weight

markers are shown to the right of lanes.

(b)

(a)

Fig. 3 Zinc-finger motifs of rat HIT-4 and ZNF597. (a) Overall protein

motifs of rat HIT-4 and ZNF597 are shown. A closed box indicates one

C2H2-zinc-finger motif (Znf1–Znf7). A box with dashed lines repre-

sents a KRAB box sequence in ZNF597. (b) The amino acid se-

quences of individual zinc-finger motifs (Znf1–Znf7) are compared.

Identical amino acids among motifs are indicated with bold letters. The

consensus sequence is shown on the bottom line.

(a) (b)

(c)

Fig. 4 Tissue distributions of HIT-4/ZNF597 mRNAs. Northern blots

of RNA extracted from cortical cultures of rat embryos (a) and various

organs of adult rats; heart, brain, spleen, lung, liver, muscle, kidney,

and testis (b). Membranes carrying total RNA (20 lg for a) or polyA+

selected RNA (5 lg for b) were probed with the 1348-bp BglII–BglII

cDNA fragment corresponding to the common zinc-finger domain of

HIT-4 and ZNF597. The positions of ribosomal RNA (18S and 28S)

are marked with arrowheads. (c) RT-PCR was performed with the

forward primers specific for exon 1A and exon 1B and the reverse

primer common for HIT-4 and ZNF597 sequences. cDNA was syn-

thesized from either cortex RNA (brain) or testis RNA and amplified in

the presence of the above primers with 16, 18, 20, 22, and 24 tem-

perature cycles. The theoretical size of PCR product for HIT-4 mRNA

is 183-bp long while that for ZNF597 mRNA is 251-bp long. Positive

controls (Posi) represent the PCR products of testis RNA with 30 cycle

amplification. Note: We confirmed that the upper band (�250 bp) in

the Posi lane was produced only with the combination of the exon 1B

primer and the common primer, while the lower one (�180 bp) was

with that of the exon 1A primer and the common primer in an inde-

pendent experiment.

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Brain defects of HIT-4/ZNF597-knockout mice | 1039

the testis and markedly strong. Considering the length ofHIT-4 and ZNF597 cDNAs, we speculated that the 6-kbsignal represents HIT-4 mRNA and the 1.5-kb signalcorresponds to ZNF597 mRNA.

To confirm this assumption, brain or testis RNA wasreverse transcribed from the common primer for HIT-4 andZNF597 sequences and subjected to PCR using the HIT-4-specific primer (for exon 1A) and the ZNF597-specificprimer (for exon 1B) (Fig. 4c). The use of brain RNAresulted in the production of the RT-PCR product (�180 bp)alone whose size matches the length calculated from HIT-4

sequence, verifying the exon 1A usage of HIT-4 mRNA. Incontrast, RT-PCR with testis RNA gave an additional PCRproduct (�250 bp) matching the size calculated fromZNF597 sequences. The 250-bp PCR product was moreabundant than that of HIT-4 mRNA in the testis RT-PCR aswell as in the brain RT-PCR. The enrichment of the 250-bpproduct in the testis and its usage of exon 1B suggest that the1.5-kb signal on northern blot is likely to represent ZNF597mRNA (see also Discussion).

Using the in situ hybridization technique, we alsoanalyzed distributions of HIT-4 mRNA in adult rat brain.Signals for HIT-4 mRNA were distributed in all the graymatter regions of adult rat brain (Fig. 5a). As neuron-likeperikarya carrying large nuclei displayed mRNA signals inthe neocortical layers excluding layer I as well as in thepyramidal layer and granule cell layer of the hippocampus(Fig. 5a–c). In the substantia grisea centralis, there weremany cells carrying intensive signals for HIT-4 mRNA(Fig. 5d). In contrast, there was no apparent signal in thewhite matter or in the neocortical layer I. All these findingssuggest the major source of HIT-4 in the brain appears to bethe neuron.

Gene targeting of HIT-4 in miceTo explore the biological function of HIT-4/ZNF597, weattempted to disrupt the common exon for HIT-4 andZNF597 in mouse ES cells to produce knockout mice. First,we screened mouse 129 genome lambda phage library andidentified the genomic clone carrying exon 3 (Fig. 6). The 4-kb BglII fragment carrying the whole translation region wasreplaced with a 3-phosphoglycerate kinase–NEO cassetteand the end of the genomic insert was linked to the diphtheriatoxin A gene. The targeting vector was introduced to mouseES cells (TT2) and 51 clones were isolated as primarycandidates for homologous recombination. Two ES clonescarried the shorter BamHI fragment that covered thisgenomic region and was positive for a probe for NEO.One ES clone was injected into mouse blastocysts to generatechimeric mice. We succeeded in germ line transmission andobtained HIT-4 mutant offspring (data not shown).

Mating heterozygous mutant mice failed to produce anyhomozygous mutants ()/)) in five litters (data not shown).Interpreting this to mean that homozygous mutants areembryonic lethal, we dissected pregnant heterozygous damsthat had mated with heterozygous males. When we dissectedpregnant mice at E8, dams carried normal embryos of thesomatic stage and abnormal embryos of the egg cylinderstage. The ratio of normal embryos to the abnormal embryoswas 3 : 1, which matches the expectation based on Mende-lian inheritance (Table 1). At E10, however, we were notable to identify abnormal embryos but found their tracesabsorbed to the uterus wall (data not shown). These findingsuggest embryonic lethality of HIT-4 homozygous mutantmice.

(a)

(b) (c)

(d)

Fig. 5 In situ hybridization of HIT-4/ZNF597 mRNAs in rat midbrain

region. HIT-4 mRNA in brain slice covering midbrain and cerebrum

regions was hybridized with the antisense and sense RNA probes

labeled with digoxigenin, and detected with the alkaline phosphatase-

conjugated anti-digoxigenin antibody system. HIT-4 mRNA-positive

cells were densely distributed in the neocortical layers, hippocampal

neurons, and substantia grisea centralis. The areas enclosed by the

dotted line in (a) correspond to (b–d). Note: A sense RNA probe for

HIT-4 did not produce any signals or background (data not shown).

Aq, aqueduct; DG, dentate gyrus; HIP, hippocampus; MB, mammillary

body; Oc, occipital cortex; R, rhinal fissure, SC, superior colliculus;

SCG, substantia grisea centralis; sn, substantia nigra; wm, white

matter. Scale bars, 1.0 mm for (a) and 0.1 mm for (b–d).

Journal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1035–1044� 2010 The Authors

1040 | Y. Tanabe et al.

Although heterozygous mutants had no problem in matingand propagation, our preliminary observation suggested thatall of three heterozygous mice displayed ataxic or epileptic

behaviors after 6 months of age (HN, preliminaryobservations). To characterize the influence of HIT-4haplo-insufficiency on brain structures, we performedconventional histological examinations of the brain(Fig. 7). We found spongiform changes in the deep layersof cingulate cortex and dorsal striatum of HIT-4 heterozy-gous mice. Furthermore, there were degenerative neuronswith condensed nucleus and scan cytoplasm in these brainregions. In contrast, there were a few vacuoles in the uppercortical layers and ventral striatum (data not shown). Thesevacuoles often associated with immunoreactivity for 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase, a cell marker foroligodendrocytes. There was no apparent sign of neurode-generation in the cerebellum, however. We did not observethe spongiform changes in wild-type littermates fixed in thesame condition. Given these results, it is possible that thebehavioral deficits of heterozygous mice might beassociated with neurodegeneration or oligodendrocytedysfunction.

(a)

(b)

Fig. 6 Targeting vector and homologous recombination of HIT-4 gene.

(a) Restriction mapping of a genomic phage clone for HIT-4, the tar-

geting vector, and homologous recombinant are shown. The targeting

vector was constructed by replacing the BglII–BglII fragment for exon 3

(3.7 kb) with 3-phosphoglycerate kinase–neomycin-resistant cassette

(pgk–NEO) by blunt-end ligation. The diphtheria toxin A fragment (DT-

A) was then attached to 3¢-end of the insert. Note: The replacement

created a unique SpeI restriction site at the position of the cassette. H,

HindIII; G, BglII; B, BamHI; K, KpnI; N, Not I; S, SpeI. (b) DNA was

extracted from G418-resistant clones of mouse ES cells, digested with

BamHI and SpeI, and separated in a 0.5% agarose gel. Southern blots

were probed with the 3¢-end probe (a 2-kb HindIII–HindIII fragment) and

NEO probe. Both probes marked the same bands. Proper homologous

recombination of the targeting vector at the 5¢-flanking region was

verified by long PCR (data not shown).

Table 1 Developmental abnormality of HIT-4 homozygous embryos

Pregnant

dams (E8)

Egg cylinder

(abnormal)

Somatic stage

(normal)

Case 1 3 7

Case 2 1 8

Case 3 3 6

Case 4 2 7

Case 5 2 6

Case 6 2 5

Total 13 39

At embryonic day 8 (E8), we dissected pregnant heterozygous dams

that had mated with heterozygous males. Under a microscope, we

classified developmental stages of embryos in the uterus.

� 2010 The AuthorsJournal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1035–1044

Brain defects of HIT-4/ZNF597-knockout mice | 1041

Discussion

In this study, we have identified the novel zinc-finger genewhose transcript is enriched in the brain. The gene encodes aprotein of 371 amino acids with seven zinc-finger motifs.The theoretical molecular weight of the open reading framematched the size of in vitro translation products from HIT-4cDNA. Subsequent BLAST search in rat genes found that thisgene shares significant nucleotide identity with ZNF597

mRNA in the protein-coding and 3¢-portions, but not in the5¢-region. ZNF597 cDNAwas originally isolated from the rattestis library (Urrutia 2003). Random sequencing projects ofexpressed sequence tag (EST) also confirm the use of thedistinct exons for 5¢-untranslated sequences for HIT-4 andZNF597 mRNAs (http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Rn&CID=64641). All three EST clones inthe cDNA libraries of the brain and sensory ganglionexclusively carries the exon 1A sequence of HIT-4 mRNA,

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Fig. 7 Cell degeneration and vacuolization

of HIT-4/ZNF597 heterozygous mutants.

We carried out neuropathological exami-

nation of HIT-4 heterozygous mutant mice

(a–c, e, g, and h) and wild-type littermates

(d and f) at PND90. Sections through hip-

pocampus (a) and cerebellum (b) of mutant

mice were examined with Kluver–Barrera

stain. Over all structures appeared to be

normal. Staining of cingulate cortex (c and

d) and the dorsal striatum (e and f) with

hematoxylin and eosin revealed pigmented

nuclei and vacuoles in mutant mice. Vacu-

oles frequently associate with regions

immunoreactive to the antibody against

2¢,3¢-cyclic nucleotide 3¢-phosphodiester-

ase in dorsal striatum (g; brown color). In

contrast, there was no apparent cell

degeneration in cerebellar layers (h).

Staining revealed no apparent differences

in sections from three animals. Scale bars,

1.0 mm for (a), 0.5 mm for (b), and 0.1 mm

in (c–h). Using long-size PCR, we fully

confirmed the homologous recombination

of exon 3 with the 3-phosphoglycerate ki-

nase–NEO cassette in the heterozygote

mutant mice (data not shown). Note: The

pathological changes were also observed in

another heterozygous mouse but not in

wild-type mice (data not shown).

Journal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1035–1044� 2010 The Authors

1042 | Y. Tanabe et al.

while all two clones in the testis library contains the exon 1Bsequence of ZNF597 mRNA. Alignment of HIT-4 andZNF597 cDNAs to rat whole genome revealed that thesegenes are produced by alternative use of the first exons in thesame gene. Accordingly, the HIT-4 gene product containsseven zinc-finger repeats, whereas ZNF597 additionallycarries a putative KRAB domain at the amino-terminal end,assuming the distinct translation initiation codon is used (Barbeet al. 2008). The UniGene expression profile by EST countsindicates that the testis and brain are the organs highlyexpressing ZNF597 mRNA (UniGene; http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.88630).Conventional EST counting is mainly based upon thesequence identity of 3¢-end of cDNA fragments. Therefore,they appeared not to distinguish mRNA sequences forZNF597 and HIT-4 and judged all to be ZNF597 mRNA.Human ZNF597 cDNA contains a poly-A tail and its totallength is 1610 bp (Barbe et al. 2008), whereas the largestcDNA clone for HIT-4 covering the entire protein cordingand poly-A tail is 5.8-kb long. The present RT-PCRexperiment indicated the usage of exon 1A in the braintranscripts and that of exon 1A and exon 1B in the testistranscripts, suggesting that 6-kb long mRNA signal innorthern blotting is likely to represent HIT-4 mRNA.Therefore, HIT-4 is a brain-enriched splicing/promotervariant of ZNF597. As there were also other sizes of RNAsignals for HIT-4/ZNF597 in cultured embryonic neurons aswell as in other tissues, unidentified exon-splicing or polyA-addition of the genome transcripts potentially exists andawaits further investigation.

Zinc-finger motifs can be classified into three categoriesbased on their amino acid composition: C2H2, C4, and GAL4(Kowalski et al. 2002). Depending on the number andpattern of the fingers, Iuchi (2001) further subclassifiedC2H2-zinc-finger proteins into three subclasses; triple-C2H2,multiple-adjacent-C2H2, and separated-paired-C2H2. HIT-4carries seven zinc-fingers adjacently that all share theconsensus amino acid sequence of the C2H2-class; Cys-X2-Cys-X12-His-X3-His (Krishna et al. 2003; Cox and McLen-don 2000). In this context, HIT-4 belongs to the subclass ofmultiple-adjacent-C2H2 including the transcription factorTFIIIA (Vrana et al. 1988). In addition to the zinc-fingermotifs of HIT-4, ZNF597 carries an amino-terminal KRABdomain. KRAB and zinc-fingers are only found together intranscription factors such as SP1, ZIF268, GLIS, ZIC2, andMZF1, which are often implicated in tissue development orcell differentiation (Urrutia 2003). Preliminary oligoDNA-systematic evolution of ligands by exponential enrichmentanalysis revealed that HIT-4 protein preferentially binds tothe (T/A)(G/A)TATA DNA motif and exhibits DNA mobilityshift of oligoDNA carrying this motif (KA, unpublisheddata). It is very likely that both ZNF597 and HIT-4 belong tothis family of transcription factors but may have distinctbiological functions. One C2H2 zinc-finger coordinates one

zinc atom which can then associates with a specific tri-nucleotide sequence (Krishna et al., 2003). Accordingly, it ispossible that HIT-4 protein, which carries seven zinc-fingermotifs, recognizes 21 nucleotides at the maximum. Takingthe neuronal presence of HIT-4 mRNA into account, wespeculate that the potential biological function of this proteinmight be associated with differentiation or development ofneurons or glial cells.

To test this hypothesis, we disrupted the common exon forZNF597 and HIT-4 with homologous recombination andproduced knockout mice. Unfortunately, we failed to obtainlive homozygous ()/)) mutant mice. However, we identifiedabnormal embryos that ceased development at the stage of eggcylinder when we examined pregnant dams at E8. Becausethe frequency of abnormal embryos followed the Mendelianrule, we concluded that homozygous mutants for ZNF597/HIT-4 are embryonic lethal. In contrast, heterozygous mutantsfor ZNF597/HIT-4 survived until adulthood. At 6 monthsof age, these heterozygous mutants developed epilepticfeatures (HN, preliminary observation). Histological analysisrevealed neurodegeneration and spongiform pathology, whichoften associated with oligodendrocytes. Neurodegenerationwas most apparent in the dorsal striatum and deep corticallayers. As ZNF597 mRNA (�1.6 kb) was undetectable in thebrain, the neuropathologic changes are presumably ascribedto the effect of haplo-insufficiency of HIT-4 expression,although its critical period responsible for the neuropathologyremains to be determined. This knockout result indicatesthe biological function of HIT-4 in the nervous system.

As HIT-4 and ZNF597 are derived from the same genomeregion, heterozygous gene deficiency of ZNF597 might alsoinfluence testis function, potentially spermatogenesis (Tascouet al. 2001; Li et al. 2007). However, pairing of male andfemale heterozygous mutants displayed no apparent problemin reproduction. Subsequent experiments will identify thetarget genes that are under transcription control of HIT-4 andZNF597, which hint at the molecular function of HIT-4 andZNF597 and their physiological roles in both brain and testis.

Acknowledgements

We thank Yoshitaka Maeda, Hiroyuki Aoki, and Kyoko Ishii for

technical assistance. This work was supported by grant-in-aids for

Scientific Research in Priority Areas (Comparative Genome) and

grants for Promotion of Niigata University Research Projects.

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