Molecular Cloning and Identification of Mouse Cklfsf2a and Cklfsf2b, Two

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The International Journal of Biochemistry & Cell Biology 38 (2006) 420–429

Molecular cloning and identiﬁcation of mouse Cklfsf2a and Cklfsf2b, two homologues of human CKLFSF2 Ting Li, Wenling Han ∗, Tian Yang, Peiguo Ding, Min Rui, Dazhen Liu, Ying Wang, Dalong Ma Peking University Center for Human Disease Genomics, 38 Xueyuan Road, Beijing 100083, China Received 13 July 2005; received in revised form 1 October 2005; accepted 6 October 2005

Abstract Human chemokine-like factor superfamily (CKLFSF) is a novel gene family comprising CKLF and CKLFSF1–8. Among them, CKLFSF2 is highly expressed in testis and may play important roles in male reproduction. Besides, it is very active during evolution and has two counterparts in mouse. For further study, we cloned the two mouse genes by EST assembly and RT-PCR methods and designated them as mouse Cklfsf2a and Cklfsf2b. Their predicted open-reading frames (ORFs) that encode 169 and 210 amino acids, respectively, were obtained; and their predicted full-length molecular sizes that are approximately 1.2 kb for mCklfsf2a and 0.9 kb for mCklfsf2b were conﬁrmed by Northern blot analysis. Mouse Cklfsf2a and Cklfsf2b show similarities with human CKLFSF2 in the expression patterns that are abundant in testis, hematopoietic and immune tissues; as well as in the chromosome localizations that neighbor CKLFSF1 and 3. Their putative protein products have 47.6 and 45.5% identities with hCKLFSF2, respectively; both of them contain four potential transmembrane regions and MARVEL domains, which are also similar with hCKLFSF2. Functionally, they all can affect the transcriptional activity of androgen receptor in PC-3 and HeLa cells, but mCklfsf2a is a repressor while mCklfsf2b and hCKLFSF2 are enhancers. Taken together, we conclude that mouse Cklfsf2a and Cklfsf2b are two homologues of human CKLFSF2. Studies on them would provide much help in further investigation of the latter. © 2005 Elsevier Ltd. All rights reserved.

Keywords: CKLFSF; Homologue; Androgen receptor; Male reproduction

1. Introduction

CKLFSF is a novel protein family that provides a Abbreviations: CKLFSF, chemokine-like factor super family; structural and functional link between chemokines and CKLF, chemokine-like factor; CKLFSF1–8, chemokine-like factor members of the transmembrane 4 super family (TM4SF). super family member 1–8; GAPDH, glyceraldehydes-3-phosphate It may have important functions across a wide range dehydrogenase; ORF, open-reading frame; MGD, mouse genome database; PCR, polymerase chain reaction; RT-PCR, reverse transcrip- of physiological and pathological processes. In human, tion PCR; TM4SF, transmembrane 4 super family; EST, expressed nine genes, CKLF and CKLFSF1–8 encode the CKLFSF sequence tag; UTR, untranslated region; MARVEL, MAL and related proteins. Among them, CKLF, CKLFSF1 and CKLFSF2 proteins for vesicle trafﬁcking and membrane link; AR, androgen are tightly linked on human chromosome 16q22.1, which receptor; ARR19, androgen receptor corepressor-19 kDa; MMTV, may suggest their close relationship (Han et al., 2003). mouse mammary tumor virus; Luc, luciferase ∗ CKLF Corresponding author. Tel.: +86 10 82802846/5031; has four RNA splicing forms: CKLF1–4. CKLF1 fax: +86 10 82801149. has a CC motif and exhibits chemotactic effects on a wide E-mail addresses: [email protected], [email protected] (W. Han). spectrum of leukocytes both in vitro and in vivo (Han et

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al., 2001). CKLF2, the full-length cDNA product encod- 2. Materials and methods ing 152 amino acids, has four putative transmembrane regions and a stimulating effect on the proliferation and 2.1. Blast-based searches and EST assembly of differentiation of C2C12 cells (Xia et al., 2002). Rat and mouse Cklfsf2a and 2b mouse Cklfs have RNA splicing forms and functions similar to those of human CKLFs (Lou et al., 2003; Rui et Using the mouse Cklf2 cDNA as a query sequence, al., 2003). Human CKLFSF1 is a complicated gene, hav- we performed BLASTN searches against the public ing at least 23 isoforms designated as CKLFSF1-v1-v23 database of ESTs (dbEST) (Banfi, Guffanti, & Borsani, whose protein products are predominantly expressed in 1998), thereby retrieving a set of related ESTs (listed in human testis and most abundant in the spermatocytes Table 1) from mouse EST databases. These ESTs were (primary and secondary) as well as in the tissue assembled into a contig from overlapping ESTs through fluid surrounding spermatogonia and spermatocytes. manual alignment, which was named mouse Cklfsf2a. The above facts indicate that they may exert critical Subsequently we used this contig as the query sequence functions in gene regulation and tissue differentiation to perform repeated BLASTN searches, whereupon we during spermatogenesis (Wang et al., 2004). Our recent found an additional set of mouse ESTs (listed in Table 1). studies show that human CKLFSF2 with four putative Following EST assembly, another contig homological to transmembrane regions can be detected in the spermato- mouse Cklfsf2a was obtained and designated as mouse gonium and can also be secreted into the tissue fluid of Cklfsf2b. human testis at the same time (Shi et al., 2005). Such evidences serve to indicate that human CKLFSF2 may 2.2. Molecular cloning of mouse Cklfsf2a and 2b have an important function in the context of germ-cell development. Based on the predicted sequences of mouse Cklfsf2a On the other hand, CKLFSF2 is active during evo- and 2b, a series of gene-specific primers were designed lution; and in mouse, it has two counterparts, mouse for the purpose of amplifying their ORFs by nested RT- Cklfsf2a and Cklfsf2b. According to our preliminary PCR. These oligonucleotides are as follows: 5-GTT studies, human CKLFSF2 can promote the transcrip- GAG AGC CAC CCT TGA ACA G-3 (P1), 5-CAC tional activity of AR (unpublished data). However, AGC TTG GCA TGG GTC C-3 (P2), 5-ACA ACC Jeong et al. (2004), reported a novel androgen receptor ATG GCA GCA CCG-3 (P3), 5-CAC AGT TAC CAC (AR) corepressor named androgen receptor corepressor- TTC CTT AAC C-3 (P4), 5-TTG AAC AGG TCA 19 kDa (ARR19), which is in fact the mouse Cklfsf2a. GGA GAC ACC AG-3 (P5), 5-GAT GGA GAA CTC They found that ARR19 represses AR transactivation in TGC TGG AAA TC-3 (P6), 5-AAC AAC CAT GGC a dose-dependent manner and that it directly associates AGC GCC-3 (P7), and 5-GCT GCG CAG TCA CCA with AR through the N-terminal and leucine zipper- TCC AG-3 (P8). Among them, P1 and P2, P5 and P6 containing regions (Jeong et al., 2004). These provide are the primers for amplifying the full-length cDNA interesting clues to explore the activities of CKLFSF2 sequence of mCklfsf2a and 2b, respectively; while P3 in male reproductive functions and its evolution across and P4, P7 and P8 are primers for amplifying the cod- different species. ing region of mCklfsf2a and 2b, respectively. The total To assist the exploration, we cloned and charac- RNA for RT-PCRwas extracted from Balb/c mouse testis terized mouse Cklfsf2a and Cklfsf2b in the present using Trizol (Life Technologies Inc., Rockville, MD) study. They exhibit similar expression patterns and according to the manufacturer’s instructions. First-strand chromosomal localizations with human CKLFSF2. On the overall amino acid level, they show 47.6 and Table 1 45.5% sequence identities with human CKLFSF2, Accession numbers of ESTs for mouse Cklfsf2a and 2b assembly respectively, and share similar characteristics with the mCKLFSF2a mCKLFSF2b latter. Luciferase assays indicated an opposite function of mCklfsf2a with hCKLFSF2 in AR transactivation BY706860 BU945940 BU605057 AV209880 as mentioned above; but mCklfsf2b was observed to BQ840031 BY706101 enhance the transcriptional activity of AR just like BU743969 AV266820 hCKLFSF2. This work is expected to be of consid- AA108901 CA464807 erable benefit in investigating the functions of human BE626217 BU962007 CKLFSF2 and studying the evolution of the gene AI449770 AV208542 BU583340 family. 中国科技论文在线 http://www.paper.edu.cn

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Fig. 1. The putative cDNA sequences of mouse Cklfsf2a (A) and Cklfsf2b (B) and the deduced amino-acid sequences of their putative products. The nucleotide sequences of the message strands are numbered from 5 to 3 and shown in small letters. The putative amino-acid sequences are shown in capital letters. Numbering is based on +1 being the first base of the ORF. The codons limiting the open-reading frame and the putative polyadenylation signals are underlined. The in-frame stop codons upstream are boxed. The shaded fragments represent the probe sequences selected for Northern blot analysis. cDNA synthesis was performed according to standard total RNA was fractionated on 1.5% (w/v) agarose– protocols using the SUPERSCRIPTTM II cDNA syn- formaldehyde gels and transferred to GeneScreen-plus thesis system (Invitrogen). One microlitre of the cDNA nylon membranes (DuPont-NEN, Boston, MA) by cap- solution was used in a standard 25 ␮l PCR amplification. illary transfer using 20× SSC (sodium chloride–sodium The DNA was denatured at 94 ◦C for 5 min followed citrate), and cross-linked using UV radiation. Specific by 30 cycles of 94 ◦C for 15 s, 56 ◦C for mCklfsf2a or portions within the sequences of mouse Cklfsf2a and 2b 58 ◦C for mCklfsf2b for 15 s, 72 ◦C for 30 s, and a final were selected through sequence alignments (as shown extension for 7 min at 72 ◦C. The secondary PCRs were in Fig. 1) and amplified via PCR using the following performed in a 25-␮l-reaction system under the condi- primers: 5-GCA CCG ATA AAG TTT CCA TTT C-3 tion of 94 ◦C for 5 min followed by 30 cycles of 94 ◦C (P9) and 5-CAA AGA CAG AAG CTT GAA CAC G-3 for 15 s, 58 ◦C for mCklfsf2a or 56 ◦C for mCklfsf2b for (P10) for mCklfsf2a and 5-GGT GTT ACG GCA TTT 15 s and 72 ◦C for 30 s, with 1 ␮l of each primary PCR ATA AG-3 (P11) and P8 for mCklfsf2b; and then cloned product as templates. The nested RT-PCR products of into the pGEM-T-easy vectors (Promega, Madison, WI). the two genes were cloned into the pGEM-T-easy vector The plasmids were linearized by NcoI (or SalI) restric- (Promega, Madison, WI). Positive clones were selected, tion endonucleases and employed as templates for anti- and after purification, the plasmids were then sequenced sense (or negative control sense) RNA probes labeling using a 3100 DNA sequencer (Applied Biosystems, Fos- with digoxigenin-UTP by in vitro transcription with SP6 ter City, CA). (or T7) RNA polymerase, respectively, using the DIG RNA Labeling Kit (SP6/T7) (Roche Diagnostic Sys- 2.3. Northern blot analysis tems) according to the manufacturer’s instructions. The membranes prepared as above were then hybridized with Total RNA from Balb/c mouse testis was isolated the anti-sense and sense probes of mouse Cklfsf2a and using Trizol (Life Technologies Inc.) according to the Cklfsf2b, respectively, and detected with the DIG Lumi- manufacturer’s instructions. Approximately 30 ␮gof nescent Detection Kit (Roche Diagnostic Systems) in 中国科技论文在线 http://www.paper.edu.cn

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accordance with the manufacturer’s directions. Chemilu- 2.7. Luciferase assays minescence was measured with LAS-1000plus imaging system (Fuji Photo Film Co. Ltd.). 2.7.1. Plasmids Plasmids for mammalian expression and in vitro 2.4. Expression pattern analysis translation of AR, mouse Cklfsf2a and Cklfsf2b were constructed using pcDNA3.1-myc-his b (−) (Invitro- Total RNA from Balb/c mouse testis, lung, brain, gen) expression vectors. MMTV-Luc reporter construct heart, spleen and thymus were isolated using Trizol (Wang et al., 2001, 2002) was kindly provided by (Life Technologies Inc.) according to the manufacturer’s Dr. C. Chang (Cancer Center, University of Rochester, instructions. Reverse transcriptions and nested-PCRs Rochester, NY). were then performed as described above to identify the expression pattern of mouse Cklfsf2a and 2b across dif- 2.7.2. Transient transfection and luciferase assays ferent tissues. Five microlitres of the secondary PCR PC-3 and HeLa cells were grown in RPMI 1640 products was analyzed in 1.0% agarose gel. Mouse medium (Life Technologies) supplemented with 10% GAPDH was ampliﬁed as an internal standard. fetal bovine serum (Hyclone). Electrotransfection were performed with the indicated amount of expression 2.5. Genomic structure and chromosome plasmids and the reporter MMTV-Luc. SV40-Renilla localization analyses luciferase reporter plasmid (pRL) (Promega) was used as an internal control (as indicated in Fig. 6). Total amounts The chromosome localization and genomic organi- of expression vectors were kept constant by adding zation of mouse Cklfsf2a and 2b were analyzed through appropriate amounts of the blank vector, pcDNA3.1- mouse BLAT search (http://genome.ucsc.edu/cgi- myc-his b (−). Transfected cells were then maintained in bin/hgBlat). The contigs NM 181990.1, NM 027022.2, RPMI 1640 medium containing 10% charcoal-stripped NM 028524.1 and NM 024217.2 were selected for fetal bovine serum (Hyclone); and 24 h later, either analysis of mouse Cklfsf1, Cklfsf2a, Cklfsf2b and 10 nM 5␣-dihydrotestosterone (DHT, Sigma) or vehi- Cklfsf3, respectively. The genomic localization and cle were added. Cells were harvested 36 h after hormone organization of human CKLFSF2 (Han et al., 2003) are addition, and the dual-luciferase reporter 1000 assay sys- herein illustrated for comparison. tem (Promega) was employed to measure the luciferase activity. 2.6. Homology analyses of the putative protein sequences 3. Results

To validate the homology between mouse Cklfsf2a 3.1. EST assembly for mouse Cklfsf2a and 2b and 2b and human CKLFSF2, a series of bioinformatics analyses were performed in order to discern their The putative cDNA and amino acid sequences for sequence identities and character similarities. The mouse Cklfsf2a are shown in Fig. 1A. The first methion- multiple-sequence analysis of mouse Cklfsf2a and 2b ine codon in the longest open-reading frame is preceded and human CKLFSF2 was performed using the Jotun by an in-frame stop codon 30 bp upstream and flanked Hein Method of MegAlign in the Lasergene software by a Kozak consensus site, which suggests that it is the program (DNASTAR Inc., Madison, WI, USA). “Struc- genuine translation start site (Kozak, 1991). The 1076 bp tural” residue weight tables and the following parameters cDNA was therefore predicted to have a 5-UTR of were used in our analysis: gap penalty, 11; gap length 124 bp, a coding region of 510 bp encoding 169 amino penalty, 3. The Protein Calculator software program acids, and a 3-UTR of 442 bp, excluding the poly (A) (http://www.scripps.edu/∼cdputnam/protcalc.html) tract. A putative polyadenylation signal (AATAAA) was was used for analysis of the physical–chemical proper- identified in the 3-UTR at position 940. The putative ties of the three protein sequences. Expasy (http://www. cDNA and amino acid sequences for mouse Cklfsf2b are cbs.dtu.dk/services/TMHMM-2.0/) was selected for the shown in Fig. 1B, which bears a formal resemblance to prediction of putative transmembrane regions in mouse Cklfsf2a. The start codon in the longest open-reading Cklfsf2a and Cklfsf2b (Krogh, Larsson, von Heijne, & frame is preceded by an in-frame stop codon 115 bp Sonnhammer, 2001). Conserved domains were searched upstream and flanked by a Kozak consensus site. The in the CDD (Conserved Domain Database) at NCBI 867 bp cDNA was predicted to have a 5-UTR of 122 bp, (http://www.ncbi.nlm.nih.gov/Structure/cdd). a coding region of 633 bp encoding 210 amino acids, 中国科技论文在线 http://www.paper.edu.cn

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anda3-UTR of 112 bp, excluding the poly (A) tract. A putative polyadenylation signal (AATAAA) was identi- ﬁed in the 3-UTR at position 854. However, the relative positions mentioned above are all based on the results of our EST assembly; conﬁrmation of the exact start sites of transcriptions will be needed to identify the precise positions.

3.2. Molecular cloning of mouse Cklfsf2a and 2b

According to the predicted sequences, we designed specific primers and amplified their coding regions from mouse testis, where the expression information of Uni- gene suggested that our objective genes were mainly expressed. DNA sequencing verified our EST assemblies and an additional isoform of mCklfsf2a encoding 101 amino acids was obtained, named as mouse Cklfsf2a-v1, while the isoform accordant with our prediction was then designated as mouse Cklfsf2a-v2. The three sequences were subsequently registered in GenBank. The accession Fig. 2. Northern blot analysis of mouse Cklfsf2a and Cklfsf2b. The total RNA was extracted from Balb/c mouse testis. Anti-sense and numbers are AY162281 for Cklfsf2a-v1, AY172740 for sense cRNA probes were used to detect the transcripts of mouse Cklfsf2a-v2 and AY162282 for Cklfsf2b, respectively. Cklfsf2a and Cklfsf2b, indicating that both of them were encoded by As Cklfsf2a-v2 is the main existing form in mouse testis the sense nucleotide strands. The mRNA of mCklfsf2b was shown to be and other tissues, ‘mouse Cklfsf2a’ mentioned in this approximately 0.9 kb long; while mCklfsf2a was indicated to have two paper refers to mouse Cklfsf2a-v2. transcripts different in size, one being about 1.2 and the other 2.5 kb.

3.3. Northern blot analysis Balb/c mouse through RT-PCR. The electrophoresis of the PCR products indicated that mCklfsf2a To identify the molecular sizes of the transcripts of was expressed in testis, spleen and thymus; while mouse Cklfsf2a and 2b, we performed Northern blot mCklfsf2b was expressed in testis, lung and thymus analysis using the total RNA isolated from Balb/c mouse (Fig. 3). testis. Besides further confirming the high expression level of mouse Cklfsf2a and 2b in testis, the results 3.5. Genomic structure and chromosome demonstrated that the proteins of mouse Cklfsf2a and localization 2b were both encoded by the sense nucleotide strands. The mRNA of mCklfsf2b was shown to be approx- To confirm that mouse Cklfsf2a and Cklfsf2b are imately 0.9 kb long; while mCklfsf2a was indicated two homologues of human CKLFSF2, we compared to have two transcripts different in size, one being mouse Cklfsf2a and 2b with human CKLFSF2 through about 1.2 and the other 2.5 kb (Fig. 2). We obtained means of a bioinformatics strategy. The chromosome the cDNA sequence of mCklfsf2a corresponding to localization analyses demonstrate that they locate on the transcript of 1.2 kb. To identify the larger form human chromosome 16 and mouse chromosome 8, of 2.5 kb, we performed 5LACE and got chimeric respectively, and neighbor CKLFSF1 and CKLFSF3 transcripts of mouse Cklfsf2a and Cklfsf1 (data not on their respective chromosomes. Mouse Cklfsf2a and shown), which may result from their partially over- Cklfsf2b are closely linked in a head-to-head arrange- lapped localizations on mouse chromosome 8 (as illus- ment, with approximately 29 kb separating their 5 ends trated in Fig. 4A) and be responsible for this larger (Fig. 4A). Moreover, human CKLFSF2 and mouse band. Cklfsf2b are organized in a similar manner, each consisting of four exons separated by three introns; while mouse 3.4. Expression pattern on mRNA level Cklfsf2a comprises five exons and four introns (Fig. 4B). The sequences of intron–exon boundaries all match We subsequently studied the distribution of mouse the consensus sequence of eukaryotic splice junctions Cklfsf2a and 2b mRNA across different tissues in (Table 2). 中国科技论文在线 http://www.paper.edu.cn

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Fig. 3. Expression pattern of mouse Cklfsf2a and 2b. The distribution of mouse Cklfsf2a and 2b mRNA across different tissues in Balb/c mouse was detected with RT-PCR. The electrophoresis indicated that mCklfsf2a was expressed in testis, spleen and thymus; while mCklfsf2b was expressed in testis, lung and thymus. This representative set of results was repeated twice.

3.6. Sequence identities and character similarities As shown in Fig. 5A, these three proteins share between mouse Cklfsf2a and 2b and human many amino acids in common. The identities between CKLFSF2 on protein level human CKLFSF2 and mouse Cklfsf2a and 2b are 47.6 and 45.5%, respectively (as shown in Fig. 5B). To investigate the sequence identities between human Based on the degree of amino-acid sequence iden- CKLFSF2 and mouse Cklfsf2a and 2b, we analyzed tity we obtained the corresponding phylogenetic tree them in silico. Alignment of the three amino acid (Fig. 5C), which illustrates the degree of kinship they sequences indicated a high degree of conservation. possess.

Fig. 4. Sketch maps of the chromosome localizations (A) and genomic organizations (B) of human CKLFSF2, and mouse Cklfsf2a and 2b. (A) They locate on human chromosome 16 and mouse chromosome 8, respectively, and neighbor CKLFSF1 and CKLFSF3 on their respective chromosomes. Mouse Cklfsf2a and Cklfsf2b are closely linked in a head-to-head arrangement, with approximately 29 kb separating their 5 ends. (B) Human CKLFSF2 and mouse Cklfsf2b are organized in a similar manner, each consisting of four exons separated by three introns; while mouse Cklfsf2a comprises ﬁve exons and four introns. Boxes labeled with 1–5 in (B) represent the exons. 中国科技论文在线 http://www.paper.edu.cn

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Table 2 Sequences of the intron–exon junctions of mouse Cklfsf2a and 2b Number Exon size (bp) 5 splice doner Intron size (bp) 3 splice acceptor mCklfsf2a 1 277 TCTTTGgtatac 101 acccagGGCTGC 2 159 ATGGCGgtaagt 8690 tttcagGATATC 3 102 GCTATGgtaaag 589 tcctagATCCTC 4 83 AAGGAAgtatgt 1676 ttctagGTGGTA 5 456 mCklfsf2b 1 262 ATTATGgtaaac 110 attcagATCCTC 2 159 ATGATGgtaagt 6996 tttcagGATCTG 3 102 GGTATGgtaagg 549 tcctagATCCTC 4 342

Exons and introns are interrupted with the GT–AG boundary consensus sequences. Exon sequences are indicated by uppercase; intron sequences are indicated by lowercase.

Comparison of the physical–chemical characteris- all predicted to have four transmembrane regions tics of mouse Cklfsf2a and 2b and human CKLFSF2 and are all MARVEL domain-containing proteins protein sequences suggests that there are many com- by conserved domain searches at NCBI (Table 3). parable features among them, for example, their iso- These similarities support the proposition that mouse electric points are all on the basic side and are Cklfsf2a and Cklfsf2b are homologues of human 9.70, 9.54 and 9.89, respectively. Besides, they are CKLFSF2.

Fig. 5. Sequence alignment of human CKLFSF2 and mouse Cklfsf2a and 2b. (A) Amino-acid sequence alignment of human CKLFSF2 and mouse Cklfsf2a and 2b. The shaded areas indicate matching residues. (B) Sequence comparison of human CKLFSF2 and mouse Cklfsf2a and 2b. The numbers represent the percentage of sequence identity. (C) Phylogenetic analysis of human CKLFSF2 and mouse Cklfsf2a and 2b. 中国科技论文在线 http://www.paper.edu.cn

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Table 3 Transmembrane regions and MARVEL domains of mouse Cklfsf2a and 2b and human CKLFSF2 Transmembrane regions MARVEL domain Amino acids

1st 2nd 3rd 4th

mCklfsf2a 40–62 72–94 107–126 136–158 40–162 169 mCklfsf2b 45–62 66–88 95–117 127–149 35–151 210 hCKLFSF2 94–111 116–135 142–166 179–198 82–198 248

Both the amino and carboxyl terminal regions are predicted to be located on the cytoplasmic side of the cell membrane.

3.7. Effects of mouse Cklfsf2a and Cklfsf2b on AR 4. Discussion transactivition The human chemokine-like factor super family rep- Human prostate cancer PC-3 cell line, which is an resents a novel gene family, whose members disperse AR-irresponsible cell line, was transiently transfected on three distinct chromosomes. CKLF and CKLFSF1–4 with MMTV-Luc reporter construct, AR expression vec- form a gene cluster on chromosome 16q22.1; CKLFSF5 tor, and increasing amounts of mCklfsf2a or mCklfsf2b is located on chromosome 14q11.2; and CKLFSF6–8 expression plasmids. The total plasmid amount was kept form another gene cluster on chromosome 3p23 (Han constant with blank vector. As shown in Fig. 6A and et al., 2003). Among them, CKLFSF3–8 are relatively C, mCklsf2a repressed, but mCklfsf2b enhanced DHT- more conserved, while CKLF, CKLFSF1 and CKLFSF2 mediated AR transactivation, both in dose-dependent are quite active during evolution. The latter three genes manners. Similar results were also observed in human are tightly linked and form a subfamily. Some regulatory cervix adenocarcinoma HeLa cells (Fig. 6B and D). elements of them are mutually overlapping; for example,

Fig. 6. Coexpression of mouse Cklfsf2a or Cklfsf2b affects the transcriptional activity of AR. PC-3 (A and C) and HeLa (B and D) cells were transiently cotransfected with 0.2 ␮g AR expression plasmid, 2.5 ␮g MMTV-Luc reporter, and increasing amounts of mouse Cklfsf2a (A and B) or Cklfsf2b (C and D) encoding plasmid. SV40-Renilla luciferase reporter plasmid was used as an internal control. Total amounts of expression vectors were kept constant by adding appropriate amounts of the blank vector. The luciferase activity of AR without coregulator in the presence of DHT was set as 100. All values represent the mean ± S.E. of at least three independent experiments. 中国科技论文在线 http://www.paper.edu.cn

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CKLFSF1 and CKLFSF2 each have functional promot- exists in testis with both the cytoplasmic and secretory ers located in their respective upstream genes, CKLF and forms (Shi et al., 2005) and has been suggested to show CKLFSF1 (Xu et al., 2004; Xu, Yang, Gao, Shi, & Ma, a stimulative effect on AR transactivation (unpublished 2005). These indicate that the three genes are likely to paper). Luciferase assays indicated that mouse Cklfsf2b derive from the early duplication of a common ancestor was also an enhancer of AR while mouse Cklfsf2a acted (Cornel, Magalie, Daniel, & Francois, 2001). as an AR repressor just as reported by Jeong et al. However, this subfamily is found to contain four (2004). It is interesting to find that homologues can exert genes—Cklf, Cklfsf1, Cklfsf2a and Cklfsf2b—in mouse. absolutely opposite functions in the same system. This In this report we have testified that mouse Cklfsf2a and will help to unravel the molecular mechanism involving Cklfsf2b are the homologues of human CKLFSF2.As in male reproduction and is expected to be of consid- viewed from no matter the chromosome localization, erable benefit in studying the evolution of this gene genomic structure or the characteristics of amino acid family. sequence, mouse Cklfsf2b can be certainly identified as ARR19 was reported to be expressed only in male a homologue of human CKLFSF2. While for mouse reproductive organs such as testis and prostate (Jeong Cklfsf2a, despite some differences of its genomic orga- et al., 2004). But in our study, we found that besides nization from that of human CKLFSF2, we ultimately testis, mouse Cklfsf2a and 2b were also expressed in confirm it to be another homologue of the latter as hematopoietic and immune tissues, such as spleen and its amino acid sequence has much higher identity with thymus. Coincidently, human CKLFSF2 is moderately human CKLFSF2 than with other proteins of this fam- expressed in bone marrow and leukocytes (Shi et al., ily in human (data not shown). Mouse Cklfsf2a and 2b 2005), which are also related to hematopoiesis and could be produced by a second gene duplication as evi- immunity. It therefore can be speculated that these genes denced by their linked structure, high level of sequence would also display some functions during these two pro- identity and comparably high expression in mouse testis cesses. and hematopoietic and immune tissues (Jianming, Su- The identification of the MARVEL domain in mouse Qing, Elizabeth, & Jeffrey 1997; Sossey-Alaoui, Head, Cklfsf2a and 2b suggests that they could be related Nowak, & Cowell, 2003). to the machinery of membrane apposition events, such There are several homologous nucleotide fragments as transport vesicle biogenesis or tight-junction regula- shared by mouse Cklf, Cklfsf1, Cklfsf2a and Cklfsf2b, tion. This could aid the design of further experimental which further indicates that they may derive from a work to investigate their associations with specialized common ancestral gene. At the same time this may pro- membranes and to identify their exact roles in the produce some confusion in mapping their exact localization cess of membrane apposition (Sanchez-Pulido, Martin- on chromosome. Analyzing their chromosome localiza- Belmonte, Valencia, & Alonso, 2002). tion with different contigs will give somewhat different results. For that reason the localization of mouse Cklfsf2a Acknowledgements and 2b described in this report is not in complete agree- ment with the situation mentioned in our previous paper This work was supported by Foundation for the (Han et al., 2003). Further investigation is necessary for Author of National Excellent Doctoral Dissertation these genes to be mapped precisely. On the other hand, of PR China (FANEDD) (200257), the National Nat- the partially overlapped localization of mouse Cklfsf1 ural Science Foundation (30271203) and the Chi- and Cklfsf2a on chromosome may produce chimeric nese High Tech Program (863) (2003AA215022 and transcripts, which have been confirmed by 5 -LACE 2002BA711A01). We also thank Dr. C. Chang (Can- experiment (data not shown) and may explain the 2.5 kb cer Center, University of Rochester, Rochester, NY) for band present in the Northern blot analysis of mouse providing AR and MMTV-Luc reporter construct. Cklfsf2a. Such circumstance was previously reported on another two genes tightly linked also on mouse chro- References mosome 8, Scyd1 and Scya17, which encode CX3C chemokine fractalkine and CC chemokine TARCrespec- Banfi, S., Guffanti, A., & Borsani, G. (1998). How to get the best of tively and generate a chimeric molecule fracTARC by dbEST. Trends in Genetics, 14(2), 80–81. alternative splicing (Hiroyama et al., 2001). 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