Subunit Genes Β Proteasome Cluster and Identification of Five New

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Analysis of a 26-kb Region Linked to the Mhc in Zebrafish: Genomic Organization of the Proteasome Component β/Transporter Associated with Antigen Processing-2 Gene This information is current as Cluster and Identification of Five New of October 2, 2021. Proteasome β Subunit Genes Brent W. Murray, Holger Sültmann and Jan Klein J Immunol 1999; 163:2657-2666; ; http://www.jimmunol.org/content/163/5/2657 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Analysis of a 26-kb Region Linked to the Mhc in Zebraﬁsh: Genomic Organization of the Proteasome Component ␤/Transporter Associated with Antigen Processing-2 Gene Cluster and Identiﬁcation of Five New Proteasome ␤ Subunit Genes

Brent W. Murray, Holger Su¨ltmann, and Jan Klein1

Sequencing of zebrafish (Danio rerio) bacterial artificial chromosome and P1 artificial chromosome genomic clone fragments and of cDNA clones has led to the identification of five new loci coding for ␤ subunits of proteasomes (PSMB). Together with the four genes identified previously, nine PSMB genes have now been defined in the zebrafish. Six of the nine genes reside in the zebrafish Downloaded from MHC (Mhc) class I region, four of them reside in a single cluster closely associated with TAP2 on a 26-kb long genomic fragment, and two reside at some distance from the fragment. In addition to homologues of the human genes PSMB5 through PSMB9, two new genes, PSMB11 and PSMB12, have been found for which there are no known corresponding genes in humans. The new genes reside in the PSMB cluster in the Mhc. Homology and promoter region analysis suggest that the Mhc-associated genes might be inducible by IFN-␥. The zebrafish class I region contains representatives of three phylogenetically distinguishable groups of PSMB

genes, X, Y, and Z. It is proposed that these genes were present in the ancestral PSMB region before Mhc class I genes became http://www.jimmunol.org/ associated with it. The Journal of Immunology, 1999, 163: 2657–2666.

roteasomes are self-compartmentalizing multicatalytic exhibit an altered proteolytic activity, producing peptides that are sim- protease complexes that play a key role in protein degra- ilar to those presented by the MHC (Mhc) class I molecules (6, 7). P dation throughout the cell (1, 2). A feature common to all The TAP molecule is part of a large superfamily of ATP-bind- proteasomes is the central core, the 20S unit, in which the proteo- ing cassette (ABC) membrane bound transporters (8). In mam- lytic activity of the complex resides (1). The 20S proteasome is a mals, TAP molecules transport antigenic peptides produced in the barrel-shaped molecule, made up of four stacked rings arranged in cytosol, preferentially those destined for binding to class I mole- an ␣␤␤␣ orientation. In eukaryotes, both the ␣ and the ␤ rings are cules, into the lumen of the rough endoplasmic reticulum (9). The by guest on October 2, 2021 comprised of seven unique proteasome component (PSM)2 sub- TAP molecule is comprised of two noncovalently associated subunits (3). Of these, only three of the ␤ subunits appear to be cat- units, TAP1 and TAP2. The genes coding for both of these sub- alytically active, and these interact with the other ␤ subunits to units are located in the mammalian Mhc, where they are tightly form a proteolytic pocket in the center of the ring structure (3, 4). linked with the PSMB8 and PSMB9 genes (10). The PSMB8, In mammals, a second 20S proteasome, the immunoproteasome, TAP1, PSMB9, and TAP2 genes of the cluster occur in the follow- has been identified (5). It differs from the “general housekeeping” ing orientation: 4333. Evidence suggests that the PSMB8 20S proteasome at the three catalytically active ␤ subunits. Upon and TAP1 genes are coregulated from a shared bidirectional induction by IFN-␥, three additional proteasome component ␤ promoter (11, 12). ϭ (PSMB) subunits are expressed, PSMB8 ( low molecular mass Proteasome and ABC transporter genes have been used to pro- ϭ ϭ protein 7 (LMP7)), PSMB9 ( LMP2), and PSMB10 ( MECL1). vide evidence in support of the hypothesis that two rounds of chro- These subunits replace the constitutively expressed housekeeping mosome duplication, presumably preceding the emergence of ϭ ϭ ␦ ϭ subunits PSMB5 ( X, MB1), PSMB6 ( Y, ), and PSMB7 ( Z, jawed vertebrates (13–17), were critical for the appearance of the MC14), respectively, during formation of the newly synthesized adaptive immune system (15). PSMB and ABC transporter genes proteasome. The resulting immunoproteasome has been shown to are part of paralogous genomic regions that are central to this hypothesis. In humans, the PSMB/TAP gene cluster is linked to the HLA complex located in the 6p21.3 region. Genes paralogous to Max-Planck-Institut fu¨r Biologie, Abt. Immungenetik, Tu¨bingen, Germany several HLA complex genes, including PSMB7, which codes for a Received for publication March 24, 1999. Accepted for publication June 11, 1999. subunit that is replaced by the PSMB10 subunit in the immuno- The costs of publication of this article were defrayed in part by the payment of page proteasome, and the ABC2 gene, are present in the q33–34 region charges. This article must therefore be hereby marked advertisement in accordance on chromosome 9 (15, 16). Kasahara and coworkers (15) hypoth- with 18 U.S.C. Section 1734 solely to indicate this fact. esized that the preduplication ancestor of the 6p21.3 region con- 1 Address correspondence and reprint requests to Dr. Jan Klein, Max-Planck-Institut fu¨r Biologie, Abt. Immungenetik, Corrensstrasse 42, D-72076, Tu¨bingen, Germany. tained three tandemly arranged PSMB housekeeping genes, pre- E-mail address: [email protected] PSMB5, pre-PSMB6, and pre-PSMB7. Upon block duplication 2 Abbreviations used in this paper: PSM, proteasome component; AA, amino acid; (presumably chromosomal), the three genes associated with the ABC, ATP-binding cassette; BAC, bacterial artificial chromosome; LMP, low mo- region destined to become the Mhc evolved into the PSMB8, lecular mass protein; PAC, P1 artificial chromosome; PSMB, proteasome component ␤; UTR, untranslated region; C/EBP, CCAAT/enhancer-binding protein; IRF, IFN PSMB9, and PSMB10 genes. The PSMB10 gene was subsequently regulatory factor. translocated to another chromosome. In the 9q33–34 paralogous

Table I. PCR primers used for cDNA analysis

Primer Sequence Gene Orientationa Relative Locationb

BM-a37-7F1 ACC ATT CTT gCA gTC AAg TTC PSMB11 F 2–8 BM-a37-7F2 gTC ATC ATT ggg TCT gAC TC PSMB11 F 12–18 BMpr5-F3 C AgA CAT CAT CCA TAC TCT CAA g PSMB11 F Ϫ15 to Ϫ7 BMpr5-F4 g TCT gAC TCC AgA gCT TCC ATg PSMB11 F 15–22 BM-b18-3R1 gC ATC AgC CAg TgA TCC AgC PSMB11 R 53–47 BM-b18-3R2 AA TAT TCg ATC gTg CAC CTg PSMB11 R 43–37

Dare*Z1-E3F1 gT gCC AAg ATT CAC TAC ATT gCA PSMB12 F 32–39 Dare*Z1-E4F2 g gAg AAg ACA ACA gAT ATg CTg TC PSMB12 F 53–61 Dare*Z1-E5R2 T gTA CAA gTg ACT gCC AgT gCA g PSMB12 R 115–106 Dare*Z1-E7R1 TTg gAT CgC ATC ACT TAC CA g Ag PSMB12 R 163–156 Dare*LMP2.F1 TC CAT gAC AAg ATC TA C TgT gC PSMB9A F 38–45 Dare*LMP2.F2 gAC gCT CAA ACT ATT gCT gAg PSMB9A F 52–58 Dare*LMP2.R1 CAg AgTgAg AgC ATT CAC TAC PSMB9A R 164–158 Dare*LMP2.R2 gT CAg CAA ACC ACT CAA TgT Tg PSMB9A R 123–116 Dare*LMP2.R3 CAg AgA CAg ACT gTT gAT CAC PSMB9B R 164–158 Dare*LMP2.R4 C ATC TTT gTC AgT AgT ATC CAC PSMB9A R 185–178 Dare*LMP2.R5 T CTC gCT gTC AAT ggT AAC gAg PSMB9B R 185–178 Dare*PSMB7.R1 CAT CgC TgC Cag AgA TCC AgA C PSMB7 R 138–131 Dare*X.F1 gTA TgC ggA gAC CTg TTC gCT A PSMB5 F 123–130 Downloaded from Dare*X.F2 gAC TTg ACC ATA gAT gAg gCT Tg PSMB5 F 148–155 ␭GT10-F1 AgCAA gTTCA gCCTg gTTAAg ␭ Vector Not applicable Not applicable ␭GT10-R1 CTTAT gAgTA TTTCT TCCAg ggTA ␭ Vector Not applicable Not applicable

a An F or R indicates the 3Ј end of the primer faces forward or reverse relative to the reading frame, respectively. b The position of the primer, 5Ј–3Ј, is given relative to the amino acid positions given in Fig. 1. http://www.jimmunol.org/ region, the PSMB7 is postulated to be the sole remainder of the and a class I gene present on a single BAC clone (B. Murray, V. alternate duplicated PSMB three-gene cluster. Michalova´, H. Su¨ltmann, and J. Klein, manuscript in preparation). To test the hypothesis of block duplication of the proposed The aim of the present study was to determine the composition paralogous regions, Hughes (18) conducted a phylogenetic analy- and organization of the Mhc-linked PSMB and TAP2 genes in the sis of the genes involved. He found the estimated times of the zebrafish. duplication events to vary considerably among the pairs of paralogous genes. This observation is inconsistent with the block dupli- Materials and Methods cation hypothesis. In particular, the divergence of the TAP1/2 and Sequencing of BAC 7 and 716 subclones by guest on October 2, 2021 ABC2 genes appears to have occurred before the divergence of Genomic regions flanking the previously identified Dare-TAP2 and eukaryotes from bacteria. As an alternative explanation, he pro- -PSMB8 genes (20) were targeted for nucleotide sequencing. Various re- posed that there may be a selective advantage to the clustering of striction enzymes were used to construct subclone libraries of BAC clones broadly expressed genes in regions likely to have a wide range of 7 and 716 (two overlapping clones previously shown to contain Mhc class transcriptional activity. According to this hypothesis, the associa- I genes; Ref. 21). Restriction fragments were excised from 1% agarose gels (Carl Roth, Karlsruhe, Germany), extracted via the QIAEX II kit (Qiagen, tion of the PSMB and ABC transporter genes with the two pro- Hilden, Germany), and cloned into the pGEM-7Zf(ϩ) plasmid vector (Pro- posed paralogous regions has occurred purely by chance, but was mega, Mannheim, Germany). Subcloned fragments spanning the region of subsequently maintained due to selective advantage. the known Dare-TAP2 and PSMB8 genes were sequenced using the The comparative analysis of nonmammalian jawed vertebrate Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech, genomes will undoubtedly lead to a greater understanding of the Braunschweig, Germany), the LI-COR DNA sequencer 4200 (MWG-Bio- tech, Ebersberg, Germany), and fluorescently labeled primers. Sequences evolution of gene associations. Unfortunately, little is known about were compared with those in the GenBank through both FASTA nucleotide the genomic organization of any nonmammalian vertebrate. Most and BLASTx searches. advanced in this regard is the study of the zebrafish, Danio rerio Analysis of zebrafish cDNA library (17). The zebrafish, a representative of the bony fishes (Osteich- thyes), is one of the model organisms in a variety of studies, but Two rounds of PCR amplification were used to obtain full-length cDNA particularly in developmental biology (19). In this species, four sequences of clones in a zebrafish cDNA library constructed from 20 adult individuals (24). In the first round, a vector-specific and a gene-specific proteasome genes have been identified (20): two from complete primer were used; in the second round, the same vector primer and a sec- cDNA sequences, Dare-PSMB8 and -PSMB6 (formerly Dare- ond gene-specific primer located just downstream of the first were applied LMP7 and -Y, respectively), and from two partial cDNA se- (Table I). PCR amplifications were conducted using the PTC-100 program- quences, Dare-PSMB9 and -PSMB5 (formerly Dare-LMP2 and -X, mable thermal controller (MJ Research, Watertown, MA). In each case, 1 ␮ respectively). Linkage studies (20) and analysis of zebrafish l of the phage suspension was used as a template and the concentrations of reagents in a 25-␮l volume were as follows: 5 pmol of each primer, 1 genomic bacterial artificial chromosome (BAC) clones containing U Taq DNA polymerase (Amersham Pharmacia Biotech), 100 mM NaCl,

class I genes (21) have shown that the Dare-PSMB8 and -PSMB9 10 mM Tris, pH 7.8, and 1.5 mM MgCl2. The thermal profile consisted of are linked to Mhc class I genes. However, in zebrafish, Mhc class 4 min at 94°C followed by 35 cycles of 94°C for 15 s, 50°C or 55°C for I and class II are not linked (22). Genes representing four families 30 s, 72°C for 2 min, and completion at 72°C for 8 min. The PCR fragments were cloned into the pGEM-T vector (Promega) . of the human 6p21.3 and 9q33–34 paralogous groups (PSMB, TAP, RING3, and RXRB-like genes) have been found in zebrafish Sequence analysis to be linked to Mhc class I genes (20, 23). All of these and the Mhc Sequence alignments were made with the aid of the computer program class I genes are present on a 500-kb contig of BAC and P1 arti- CLUSTAL W (25) with minor improvements made by eye. Phylogenetic ficial chromosome (PAC) clones, with the PSMB, TAP, RING3, trees were constructed by the neighbor-joining method (26) from pairwise The Journal of Immunology 2659 distances estimated from amino acid (AA) alignments using both the pro- extends 720 bp from the position of the BM-a37-7F1 primer to the grams “neighbor” and “protdist” contained in the PHYLIP, version 3.5c, beginning of the polyA tail. The sequences are identical in the computer package (27) and the MEGA, version 1.02, computer program 84-bp region of overlap. The entire cDNA sequence is 855 bp (28). In each case, any positions containing indels were excluded from the analysis. The trees were bootstrapped 500 times (29). long, extends up to the beginning of the polyA tail, and includes 74 bp of the 5Ј UTR and 127 bp of the 3Ј UTR. A possible polyad- Screening of PAC library enylation signal is found at sites 103–108 of the 3Ј UTR. The AA The PAC zebrafish genomic library no. 706 was obtained from the Re- sequence of the entire polypeptide is 217 AA residues long. It is source Center/Primary Database of the German Human Genome Project, comprised of a 16-AA propeptide and a 201-AA mature protein Max-Planck Institute for Molecular Genetics (Berlin-Charlottenburg, Ger- deduced from similarity to other PSMB subunits (Fig. 2) and from many; http://www.rzpd.de). It was screened with PCR-amplified fragments the presence of the correct glycine (G)-threonine (T) motif, which of the Dare-PSMB9A and Dare-PSMB9B cDNA pGEM clones. The PCR products were isolated from a low-melting-point agarose gel, labeled with is the site of the cleavage of the N-terminal propeptide (1). 50 ␮Ci [␣-32P]dCTP by the random priming method using the Ready- To-Go kit (Amersham Pharmacia Biotech) and hybridized to the PAC fil- Dare-PSMB12 ters following the suggested protocol (Resource Center/Primary Database). The Dare-PSMB12 cDNA sequence (Fig. 1) is also based on two Genomic organization overlapping PCR-amplified fragments. The first fragment extends 462 bp from the Dare*Z1-E5R2 primer to the transcription initia- Overlapping DNA sequence fragments generated from the analysis of the tion site. The second fragment extends 723 bp from the position of subclone libraries were identified and organized with the computer program AssemblyLIGN (Eastman Kodak, Rochester, NY). For each contig, the Dare*Z1-E4F2 primer to the beginning of the polyA tail. No the exon/intron organization was deduced from the known cDNA se- nucleotide differences are found in the 132-bp overlap. No 5Ј UTR quences. Intron-specific PCRs were conducted to join the existing contigs, is detected in the 1002-bp sequence, while a 161-bp 3Ј UTR is Downloaded from confirm exon/intron boundaries, and estimate the sizes of introns. The found that contains a polyadenylation signal at sites 148–153 and PCR, cloning of amplified fragments, and DNA sequencing were per- formed as described above (primer sequences available upon request). In a polyA tail after site 161. However, analysis of the genomic se- all cases, BAC 716 DNA was used as a PCR template. quence reveals the presence of a probable initiation codon (shown in lower case and italics in Fig. 1) that is lacking in the cDNA Analysis of promoter regions sequence. Based on this initiation codon the deduced mature pro- DNA regions extending 700 bp upstream of the initiation codon of each tein is 237-AA residues long after the removal of a 44-AA residue http://www.jimmunol.org/ gene were analyzed for possible regulatory motifs. Sequences were com- long propeptide (assuming cleavage at the GT motif). pared with the TRANSFAC database (30) with the computer program TFSEARCH, version 1.3 (Y. Akiyama, TFSEARCH: Searching Transcrip- Dare-PSMB9 tion Factor Binding Sites, http://www.rwcp.or.jp/papia/). Based on a fragment of the Dare-PSMB9 gene reported previously Nomenclature (20), primers were designed to characterize the full-length cDNA This paper follows the convention for naming Mhc genes (31). Genes with sequence. Three sequences were detected: two alleles of the orig- homology to previously named genes in other organisms are given the inal locus renamed Dare-PSMB9A and a second locus identified on same name in zebrafish, (e.g., Psmb in mouse, PSMB in human and ze- the basis of a divergent sequence and denoted Dare-PSMB9B. by guest on October 2, 2021 brafish). Alleles are designated by an asterisk and a numerical code. The full-length Dare-PSMB9A*01 cDNA sequence (Fig. 1) was derived from two overlapping PCR fragments. The sequence of the Results first fragment is 594 bp long, extending from the 5Ј UTR to the Identification of new genes position of the Dare*LMP2.R1 primer. The second fragment, To identify new genes in the Mhc class I region of zebrafish, sub- which is identical in the 291-bp overlap to the first one, extends clone libraries of the overlapping zebrafish BAC clones 7 and 716 from the position of the Dare*LMP2.F2 primer to the end of the (21) were constructed. Through direct sequencing of targeted sub- shown 3Ј UTR. The Dare-PSMB9A*01 sequence is identical cloned fragments, genomic sequences were found that contained throughout its length to the previously reported fragment (20) and exons of new proteasome genes. From a TaqI subclone library, we is most likely the full-length cDNA clone of this gene. The se- recovered genomic fragments that contained possible exons with quence is 885 bp long and contains 54 bp of the 5Ј UTR and 174 sequence similarity to the mammalian proteasome genes PSMB6 bp of the 3Ј UTR. Although a polyA tail has not been found, a or PSMB9 (LMP2; Refs. 32–34). In the second subclone library, an potential polyadenylation signal is present at sites 167–172. The XhoI fragment (3.3 kb) was found to contain exons related to the sequence codes for a deduced propeptide of 19-AA residues and a proteasome genes PSMB7 and PSMB10 (35). mature protein of 199-AA residues (assuming cleavage at the GT Based on the exons detected, primers were designed (Table I) to motif). screen the zebrafish cDNA library. In addition, primers were de- An additional PCR fragment was found with high sequence sim- signed based on the partial zebrafish PSMB5 and PSMB9 gene ilarity to the PSMB9A*01 gene. The similarity extends from the sequences described previously (20) and on EST sequences with position of the Dare*LMP2.R1 primer along the entire length of similarity to PSMB7. Through the screening of the cDNA library, the 594 bp PSMB9A*01 fragment, but the sequence extends by 263 six full-length and one partial cDNA sequences were found (Fig. bp into the 5Ј UTR. Thirteen substitutions differentiate these two 1). These included sequences of two new genes, Dare-PSMB11 sequences, six in the 5Ј UTR and seven synonymous substitutions and -PSMB12, the originally described Dare-PSMB5 and Dare- in the coding region. The 3Ј UTR sequence of this cDNA sequence PSMB9 (now called PSMB9A) genes (20), a second related PSMB9 was not determined. Because of its high similarity to the Dare- gene, named Dare-PSMB9B, and the Dare-PSMB7 gene. PSMB9A*01 gene, the sequence is interpreted as being an allele of this gene and as such it is designated Dare-PSMB9A*02 (not Dare-PSMB11 shown). The cDNA sequence of Dare-PSMB11 (Fig. 1) is based on two Another full-length cDNA sequence with similarity to the Dare- overlapping amplified fragments. The first fragment extends 238 PSMB9A genes was found based on two overlapping PCR frag- bp from the position of the BM-b18-3R2 primer to the beginning ments (Fig. 1). The 5Ј part of the sequence extends 643 bp from the of the 5Ј untranslated region (UTR), while the second fragment position of the Dare*LMP2.R3 primer; the 3Ј part of the sequence 2660 PROTEASOME GENES IN ZEBRAFISH Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Nucleotide sequences of ﬁve cDNAs derived from zebraﬁsh (Dare) PSMB7, PSMB9A, PSMB9B, PSMB11, and PSMB12 genes. Sequence identity with the uppermost sequence is indicated by a dash and an indel by an asterisk. Missing coding sequence information was deduced from the genomic sequence and is indicated by lower case italicized letters. The possible polyadenylation sites in the 3Ј UTR are underlined. The Journal of Immunology 2661 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Amino acid alignment of proteasome subunits: (A) propeptide sequence and (B) mature protein sequences (used for phylogenetic analysis). Sequences can be identiﬁed from their GenBank accession numbers and the references contained therein. Proteasome subunits are from Saccharomyces cerevisiae, Sace PRE2 (M96667), PRE3 (X78991), PUP1 (X61189); Lampetra japonica, Laja PSMB6 (D87690); Danio rerio, Dare-PSMB5, -PSMB7, -PSMB9A, -PSMB9B, -PSMB11, -PSMB12 (AF155576–81), -PSMB6 (AF0323392); Xenopus laevis, Xela-PSMB6 (D87689), -PSMB8A (D44540), PSMB8B (D44549), -PSMB9 (D87687); and Homo sapiens, Hosa-PSMB5 (D29011), -PSMB6 (D29012), -PSMB7 (D38048), -PSMB8 (Z14982), -PSMB9 (U01025), -PSMB10 (X71874). The zebraﬁsh subunits linked to Mhc class I genes are underlined. Positions relative to the N terminus of the mature protein (ϩ1) are shown. At each position, identity to the consensus sequence is shown by a dash while an indel is indicated by an asterisk. 2662 PROTEASOME GENES IN ZEBRAFISH

FIGURE 3. Midpoint rooted neighbor-joining dendrogram based on protein sequence distances among proteasome subunits (mature protein) estimated by the p-distance method (28). Values placed to the left of each node indicate the percentage of times the subunits joined by the node were found in a monophyletic clade in the consensus tree of the bootstrap analysis. The zebraﬁsh subunits linked to Mhc class I genes are underlined. Other subunits in- clude Saccharomyces cerevisiae, Sace-PRE2 (M96667), -PRE3 (X78991), -PUP1 (X61189); Lampetra japonica, Laja-PSMB6 (D87690); Danio rerio, Dare-PSMB5 (AF155578), -PSMB6 (AF0323392), -PSMB7 (AF155581); Xenopus laevis, Xela-PSMB6 (D87689), -PSMB8A (D44540), -PSMB8B (D44549), -PSMB9 (D87687); and Homo sapiens, Hosa-PSMB5 (D29011), -PSMB6 (D29012), -PSMB7 (D38048), -PSMB8 (Z14982),

-PSMB9 (U01025), -PSMB10 (X71874). Downloaded from

extends 530 bp from the position of the Dare*LMP2.F2 primer. No deduced 275-AA polypeptide is comprised of a 41-AA propeptide differences are found in the 291-bp long overlap. This sequence and a 234-AA mature protein (assuming cleavage at the GT motif). has a 119-bp long 5Ј UTR, a 57-bp long 3Ј UTR, and a potential The 3Ј UTR is 100 bp long up to the start of the polyA tail and http://www.jimmunol.org/ polyadenylation signal at sites 33–38, but no polyA tail. A 227-AA contains a possible polyadenylation signal at sites 90–95. polypeptide is deduced from the coding region, and a 17-AA propeptide and a 210-AA residue long mature protein are postu- Dare-PSMB5 lated based on the conserved GT motif. A comparison with the The partial sequence of the previously reported Dare-PSMB5 gene PSMB9A*01 sequence shows low similarity in the 5Ј and 3Ј UTR (20) was extended in the 3Ј direction to conduct a phylogenetic and a slightly longer coding region containing 66 nucleotide dif- analysis that included the complete mature protein of all known ferences. The sequence divergence suggests that this sequence rep- zebrafish PSMB subunits. We amplified a 706-bp fragment that resents a second PSMB9 locus, which we designate Dare- extended from the primer Dare*X.F2 (Table I) to the end of a by guest on October 2, 2021 PSMB9B, and this suggestion is borne out by mapping studies. polyA tail (including a possible polyadenylation signal). The se- Screening of a zebrafish genomic PAC library with fragments of quence is identical with the previous (AF032391) in the 43-bp the PSMB9A and PSMB9B cDNA clones has shown the PSMB9A overlap and extends the deduced polypeptide 37 AA up to the stop and PSMB9B genes to reside on different Mhc class I gene-con- codon (Fig. 2). The complete Dare-PSMB5 cDNA sequence is taining PAC clones: the PSMB9A gene resides on BAC clones 7 1296 bp long (not shown; GenBank accession no. AF155578). and 716 and the PSMB9B gene on the PAC clone BUSMP706A2470Q3 (B. Murray, V. Michalova`, H. Su¨ltmann, and J. Klein, unpublished observations). Phylogenetic analysis Finally, the screening of the PAC clones revealed the presence Phylogenetic trees were constructed based on distance estimates of a third PSMB9-like locus on a clone (BUSMP706P02172Q3) from an AA alignment (Fig. 2). Three distance estimates were that does not contain either the PSMB9A or the PSMB9B loci. PCR used, the first based on simple proportional (p) distances (28), the amplification of this clone with PSMB9-specific primers yielded a second based on the Dayhoff PAM matrix, and the third based on product that contained parts of intron 5 and exon 6. The sequence the categories method (27). All three distance estimates resulted in is divergent from both PSMB9A and -B but more closely related to the same topology (except for the PSMB6 clade of jawed verte- PSMB9B (data not shown). On the basis of its position on the brates). For this reason, only the tree based on the p-distances is zebrafish Mhc map and its sequence divergence, we interpret the shown (Fig. 3). Bootstrap values show strong support (99–100) for sequence as being derived from a third PSMB9 locus that we des- the grouping of each of the three types of ␤ subunits with one of ignate Dare-PSMB9C. A transcript of PSMB9C was not found in the ancestral yeast subunits, Y (PRE3, PSMB6, PSMB9), Z our cDNA library. (PUP1, PSMB7, PSMB10), and X (PRE2, PSMB5, PSMB8), and for each of the PSMB clades (PSMB5–9). The positions of the Dare-PSMB7 Dare-PSMB9A and -PSMB9B subunits are consistent with previ- Five partial zebrafish cDNA sequences with similarity to the hu- ous phylogenies (20) and support the introduced nomenclature. man PSMB7 gene were recovered from the GenBank EST library The new Dare-PSMB11 and -PSMB12 subunits are clearly mem- (AA605681, AA606112, AI331717, AI332003, and AI332014). A bers of the Y (PRE3) and Z (PUP1) clades, respectively. In both consensus sequence was generated which extends 704 bp 5Ј of the cases, they are only distantly related to the other subunits and their end of the polyA tail. The primer Dare*PSMB7.R1 (Table I) was phylogenetic positions within the clades are unclear. In the Y designed to amplify the remaining 5Ј end of the cDNA sequences. clade, PSMB11 is a sister subunit to all other subunits, while PRE3 A 528-bp fragment was recovered that was identical with the ini- is a sister subunit to the PSMB6 clade. In the Z clade, PSMB12 is tial consensus in the 248-bp overlap. No 5Ј UTR or initiation a sister subunit to the PSMB7 clade. However, in no case are these codon is present in the resulting 962-bp transcript (Fig. 1). The positions supported by the bootstrap analysis (Fig. 3). The Journal of Immunology 2663 Downloaded from http://www.jimmunol.org/

FIGURE 4. Genomic organization of the TAP2, PSMB9A, PSMB11, PSMB12, and PSMB8 genes. Positions of HindIII (H), XhoI (X), SalI (S), and MluI (M) restriction sites are listed on the full map. Exons are indicated by ﬁlled boxes. The positions and relative sizes of the six contiguous (CTG) regions of DNA sequence are shown below the full map. Detailed maps of the TAP2, PSMB9A, PSMB11, PSMB12, and PSMB8 genes are given underneath the full map. Sizes of introns and exons are given in base pairs. The locations of the C/EBP-␤ and IRF transcription binding sites are indicated by asterisks and circumﬂexes, respectively. by guest on October 2, 2021

Genomic organization Analysis of promoter regions Six regions of contiguous DNA sequence (contigs), spanning a The analysis of the promoter regions for transcription factor bind- segment of about 26 kb (Fig. 4), were assembled based on the ing motifs showed many possible transcription factor binding sites sequence information derived from the analysis of subclone librar- (data not shown). Of interest here is that no SP1 sites, found in the ies and intron-specific PCRs. All contigs are joined by clones for mammalian PSMB8 and PSMB9 genes (33, 37, 38), were found in which the complete sequence was not determined. For each gene, any of the proteasome gene promoter regions searched. Further, in a detailed map of the intron/exon organization was deduced. In each promoter, a possible CCAAT/enhancer-binding protein (C/ every case, correct splice signals were found at the intron/exon EBP)-␤ (NF-IL-6) site is present (Figs. 4 and 5). In each case, the boundaries. The Dare-TAP2 gene organization was deduced from position of the transcription factor motif is given relative to the the partial zebrafish cDNA sequence (exons 8–11; Ref. 20) and initiation codon (Fig. 5). The C/EBP-␤ nuclear factor is an acti- from the salmon (Salmo salar) TAP2 gene (36). Eleven exons of vator of various acute-phase proteins (39) and is present in the similar sizes and splice site locations to salmon (36) and human mammalian PSMB10 promoters (35). Of particular interest is the (32) TAP genes were identified. The exact size of exon 1 is not presence of the IFN regulatory factor (IRF, also known as ISRE) known and the given estimate is based on the position of a me- motif in the TAP2, PSMB11, and PSMB12 promoters (Figs. 4 and thionine codon most similar to that identified as the start of trans- 5). This motif binds both the activator IRF-1 and repressor IRF-2 lation in salmon. Two other possible initiation codons exist. Six transcription factors (40) and is present in the mammalian TAP2, exons of the Dare-PSMB8 were deduced from the existing cDNA PSMB8, and PSMB10 promoters (32, 35, 41, 42), as well as the sequence (20). Only a partial sequence of exon 6 was available for bidirectional promoter of the TAP1 and PSMB9 genes (11, 12). analysis; however, based on the length of the Dare-PSMB8 cDNA The zebrafish PSMB9A and PSMB8 genes lack this element. The and the similarity of the organization to other PSMB8 genes, this initiation codons of the PSMB11 and PSMB12 genes are 159 bp is most likely the last exon of the gene. The intron/exon organi- apart. The IRF element of the PSMB11 gene is located in the first zation of the Dare-PSMB9A, -PSMB11, and -PSMB12 genes was intron of the PSMB12 gene and vice versa. deduced from the cDNA sequences reported herein. The Dare- PSMB9A and -PSMB11 genes have a very similar organization, each possessing six exons of similar size and splice site locations. Discussion The Dare-PSMB12 gene contains eight exons. Analysis of the first In this and an earlier publication (20), we have identified nine exon reveals a probable initiation codon one codon upstream of the PSMB loci in the zebrafish genome. Phylogenetically, the loci fall end of the reported cDNA sequence. into three groups, which, for convenience, we designate X, Y, and 2664 PROTEASOME GENES IN ZEBRAFISH

PSMB9 (LMP2), and TAP2 in one direction, as well as PSMB12 and PSMB8 (LMP7) in the other direction (Fig. 4). However, the TAP2 gene also has an additional IRF-binding site in its own promoter region. In humans (and other mammals tested thus far), only two PSMB loci are present in the Mhc (PSMB8 and PSMB9) within the class II region (32, 43). Both loci are of the i type; the third i-type locus (PSMB10), as well as all the other PSMB loci, are found outside of the Mhc (5). In the zebrafish, the situation is more complicated. Here, there are at least six PSMB loci in the Mhc, but not in the class II region; instead they are all in the class I region. Because the i-type PSMB genes are functionally tied to the class I and not to the class II Mhc genes (5), it can be argued that the zebrafish arrangement makes more sense, particularly because, in this spe- FIGURE 5. Consensus sequences of two transcription factor binding cies, the class I and class II loci are on different chromosomes (22). motifs, C/EBP-␤ and IRF, and sequences of the PSMB9A, PSMB11, Furthermore, in the zebrafish, the Mhc-associated loci are presum- PSMB12, and PSMB8 promoter regions with similarity to these motifs. ably all of the i type and they represent all three PSMB groups (in Nucleotide identity is shown in bold, nonidentity in lower case. For posi- mammals, the two loci in the Mhc represent the X and Y groups; tions allowing any base pair (i.e., n), the presence of a base pair is shown the i-type locus of the Z group is on a different chromosome). Four by a dash. The relative position of the motif upstream from the translation of the six Mhc-associated zebrafish PSMB loci are in a single main Downloaded from initiation codon is indicated. cluster; the other two are at a distance of ϳ60 kb (PSMB9C) and ϳ120 kb (PSMB9B) from the cluster (B. Murray, V. Michalova`, H. Su¨ltmann, and J. Klein, manuscript in preparation). The associa- Z, the symbols used originally for some of the human homologues tion of i-type PSMB genes with Mhc class I in zebrafish is in (Fig. 3). The groups are identified not only by shared substitutions, agreement with Hughes’ (18) hypothesis of a selective advantage the length of propeptide sequences (Fig. 2), and insertions/dele- to the clustering of genes that have similar broad range expression http://www.jimmunol.org/ tions (indels; group X-specific indel at site 95, group Y-specific patterns. indels at sites 109, 218, 219, and group Z-specific indels at sites In both humans and zebrafish, the PSMB clusters are associated 184–209; Fig. 2), but also by a characteristic exon-intron organi- closely with the TAP loci (TAP1 and TAP2 loci in humans and zation (six exons in genes of groups Y and X, eight exons in group TAP2 locus in the zebrafish; the TAP1 locus could not be identified Z genes, whereby the exons in genes of groups Y and X are dis- in this species thus far) and loosely with the RING3 locus (32). The tinguished by their different lengths and splice site locations; Fig. conservation of a close linkage of the TAP2 gene and the PSMB 4). In each of these groups, the human counterparts are distin- cluster between bony fish and mammals, despite genomic rearrange- guishable into two types on the basis of their expression—the con- ment, again suggests that it might have a selective advantage (18). by guest on October 2, 2021 stitutively expressed genes and genes inducible by IFN-␥ (5). For The degree of sequence divergence between the Dare-PSMB9A brevity, we refer to these as the c and i types, respectively. The and Dare-PSMB9B genes is similar to that reported for Xenopus zebrafish loci are distributed among these groups as follows: group laevis, Xela-PSMB8A (LMP7A) and Xela-PSMB8B (LMP7B) X (PSMB5, PSMB8), group Y (PSMB6, PSMB9A, PSMB9B, genes (44). Both sets of genes have highly diverged 5Ј and 3Ј PSMB9C, PSMB11), and group Z (PSMB7, PSMB12; Fig. 3). The UTRs and a similar degree of AA identity in the mature protein human c types in these three groups are PSMB5 (X), PSMB6 (Y), sequence (85% for Dare and 90% for Xela). However, segregation and PSMB7 (Z); the human i types are PSMB8 (LMP7), PSMB9 studies in Xenopus indicate that the two Xela genes may be allelic (LMP2), and PSMB10 (MECL1). The constitutiveness vs induc- (44). In contrast, the Dare genes reside at different loci. ibility of the zebrafish PSMB genes has not been tested (if for no The main PSMB cluster, which extends over ϳ18 kb, consists of other reason than the zebrafish IFN-␥ has not been identified as the PSMB9A, PSMB11, PSMB12, and PSMB8 loci, arranged in yet), but a tentative assignment of the types can be made by two this order in the following orientation 4433(Fig. 4). The criteria—sequence homology and the presence or absence of rel- two PSMB loci outside the main cluster are apparently the result of evant sequence elements (transcription factor binding sites) in the a duplication of the PSMB9 locus. Because phylogenetically the genes’ promoter regions (Fig. 5). Specifically, we interpret the PSMB9B and PSMB9C loci appear to be more closely related to presence of the NF-IL-6 and IRF binding sites as an indication that each other than either of them is to PSMB9A, they are presumably the associated gene might be of the i type. By these criteria, we derived from a common ancestor that had a common ancestor with classify the zebrafish PSMB8, PSMB9A, and PSMB9B loci as be- the PSMB9A locus. Whether the B and C loci are functional is ing most probably of the i type, the PSMB11 and PSMB12 loci as unclear at this time; in a cDNA library only the transcript of the B being probably of the i type (all these loci are in the zebrafish Mhc locus has been found. However, the PSMB9B locus is apparently class I region), and the PSMB5, PSMB6 (two loci that are not in the present in some haplotypes and absent in others (B. Murray, V. Mhc region), and PSMB7 loci as being of the c type. Michalova`, H. Su¨ltmann, and J. Klein, manuscript in preparation). The location of the IRF-binding sites in the zebrafish PSMB The two extra loci in the main zebrafish PSMB cluster, PSMB11 clusters may not be fortuitous. In humans, an IRF site is positioned and PSMB12, are of special interest because of their location and in the region between the TAP1 and the PSMB9 (LMP2) genes (32, their phylogenetic relationships. The PSMB11 locus is a member 43) and is believed to regulate the bidirectional expression of both of the Y group, which can be divided into two subgroups repre- these genes, which are arranged in a head-to-head orientation (11, sented in humans by the PSMB6 and PSMB9 loci. Because the 12). A similar head-to-head arrangement exists in zebrafish, except zebrafish genome contains close relatives of these two loci (Dare- in this case both the PSMB11 and PSMB12 promoters possess a PSMB6 and Dare-PSMB9, respectively) and on the phylogenetic separate IRF-binding site. The central position of these promoters tree the branch leading to Dare-PSMB11 splits off before the may influence the expression of four or five genes, PSMB11, PSMB6 and PSMB9 branches split from each other (Fig. 3), The Journal of Immunology 2665 the PSMB11 gene appears to have arisen before the divergence combination with differential peptide binding by different families of the bony fish and mammalian lineages. What has happened to of Mhc class I molecules. The various immunoproteasomes could PSMB11 in mammals is unclear at this time: it may have been lost have coevolved with separate Mhc molecules, and the apparent or it may be present but unidentified. However, it is certain that if presence of the PSMB9B gene in some haplotypes but not in others it is present, it is not located in the Mhc class II region because the may be a reflection of this evolution. In humans, immunoprotea- entire region has now been sequenced and no PSMB11 homologue somes have been shown to produce peptides with hydrophobic or has been found. The zebrafish PSMB cluster may have been as- basic carboxyl termini (6), which are well suited for binding in the sembled from genes that were originally on different chromosomes C-terminal anchor pocket of the HLA class I molecules (7). Spec- or it may have arisen by in situ duplication. Taking into account ulatively, each immunoproteasome may produce a set of peptides the closeness of the loci and their orientation in the cluster, the with a specific type of C termini. This hypothesis predicts the latter explanation is the more parsimonious of the two. Therefore, existence of corresponding class I molecules specialized for bind- we propose that the ancient PSMB cluster in the part of the chro- ing the products of different immunoproteasomes. In an attempt to mosome that later became the Mhc class I region contained the test the binding specificity of fish class I molecules, Okamura et al. ancestors of the PSMB6, PSMB9, and PSMB11 genes. (48) compared the AA variation at the C-terminal anchor pocket of It probably also contained the ancestor of the PSMB12 gene. carp class I genes with the four conserved residues (Tyr84, Thr143, The zebrafish PSMB12 gene clusters with the PSMB7 (Z) genes in Lys146, and Trp147) of the mammalian classic class I molecules. a clade that also contains the human PSMB10 (MECL1) gene. The The comparison revealed conservation of between one and three of Dare-PSMB12 gene is not the orthologue of the Hosa-PSMB7 these residues in carp molecules, indicating a possible expansion gene because a Dare-PSMB7 gene exists. It is also unlikely that the of peptide binding specificity (48). In the zebrafish, three class I zebrafish PSMB12 gene is an orthologue of the human PSMB10 loci have been described (49), all of which possess the same three Downloaded from gene because the genetic distance between the two sequences is conserved residues (Thr143, Lys146, and Trp147), as found in the much greater than that between any human-zebrafish PSMB or- most conserved carp gene. However, the full range of class I genes thologues. Further, the inclusion of nurse shark (Ginglymostoma in zebrafish has yet to be described. Additional zebrafish class I cirratum) and hagfish (Eptatretus stouti) PSMB7-like subunits (M. genes have been identified (H. Su¨ltmann, B. Murray, and J. Klein, Kasahara, unpublished observations) to this analysis shifts the po- unpublished observations), and the degree of allelic diversity is sition of the Dare-PSMB12 subunit outside the PSMB7/10 clade being investigated at these loci. The above hypothesis predicts the http://www.jimmunol.org/ (not shown). Therefore, it is most likely that the Dare-PSMB12 presence of class I molecules with diverse C-terminal anchor gene is derived either from a gene that was also the ancestor of pocket motifs in the zebrafish. PSMB7 or from a gene that was the ancestor of both PSMB7 and PSMB10. It is not possible at present to decide between these two Acknowledgments alternatives (the bootstrap support for the alternative depicted in We thank Dr. Colm O’hUigin for critical reading of the manuscript, as well Fig. 3 is too low to carry any weight). In either case, the PSMB12 as Ms. Jane Kraushaar for editorial and Ms. Sabine Jantschek for technical gene appears to have diverged from PSMB7 or both PSMB7 and assistance. We also thank Ms. Vera Michalova`for sharing the PAC clones, PSMB10 before the divergence of the bony fish and mammalian and Dr. Masanori Kasahara for providing access to the unpublished nurse by guest on October 2, 2021 lineages and the loss of PSMB12 in the latter (if mammals really shark and hagfish PSMB7-like subunit sequences. lack this gene) must have been a secondary event. References Because the X, Y, and Z groups of the PSMB genes each contain a yeast gene, the groups must have separated from one another 1. Baumeister, W., J. Walz, F. Zu¨hl, and E. Seemu¨ller. 1998. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92:367. before the divergence of lineages leading to fungi and Metazoa 2. Tanaka, K. 1998. Proteasomes: structure and biology. J. Biochem. 123:195. (Fig. 3; Ref. 45). 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