Evolutionary conservation of zebrafish linkage group 14 with frequently deleted regions of human 5 in myeloid malignancies

Ting Xi Liu*, Yi Zhou†, John P. Kanki*, Min Deng*, Jennifer Rhodes*, Hong Wei Yang*, Xiao Ming Sheng†, Leonard I. Zon†‡, and A. Thomas Look*§

*Department of Pediatric Oncology, Dana-Farber Cancer Institute, †Department of Medicine, and ‡Howard Hughes Medical Institute, Children’s Hospital, Boston, MA 02115

Edited by Janet D. Rowley, The University of Chicago Medical Center, Chicago, IL, and approved January 8, 2002 (received for review October 18, 2001) Recurring interstitial loss of all or part of the long arm of chromo- Ϸ1.5 Mb, flanked proximally by D5S479 and distally by D5S500 some 5, del(5q), is a hallmark of myelodysplastic syndrome and (2, 4). The distal CDR was identified in chromosome bands acute myeloid leukemia. Although the affected by these 5q32-q33. Flanked by the ADRB2 and IL12B genes, it spans up changes have not been identified, two critically deleted regions to 3 Mb and is found primarily in patients with 5qϪ syndrome, (CDRs) are well established. We have identified 76 zebrafish cDNAs a distinct type of MDS characterized by refractory anemia, a orthologous to genes located in these 5q CDRs. Radiation hybrid favorable prognosis, a low rate of transformation to acute mapping revealed that 33 of the 76 zebrafish orthologs are clus- leukemia, and deletion of the 5q region as the sole cytogenetic tered in a genomic region on linkage group 14 (LG14). Fifteen abnormality (14–18). others are located on LG21, and two on LG10. Although there are Despite reports of increasingly smaller CDRs, attempts to large blocks of conserved syntenies, the order between identify tumor suppressor genes in these regions have been human and zebrafish is extensively inverted and transposed. Thus, unrewarding (1, 19). Studies to identify mutations within re- intrachromosomal rearrangements and inversions appear to have tained alleles of genes reduced to hemizygosity, as well as occurred more frequently than translocations during evolution potential submicroscopic homozygous deletions in known from a common chordate ancestor. Interestingly, of the 33 or- CDRs, also have been unproductive (5), suggesting either that thologs located on LG14, three have duplicates on LG21, suggest- haploinsufficiency is adequate to promote myelodysplasia or that ing that the duplication event occurred early in the evolution of the retained tumor suppressor allele has undergone epigenetic teleosts. Murine orthologs of human 5q CDR genes are distributed inactivation (20, 21). A number of putative tumor suppressor among three , 18, 11, and 13. The order of genes gene candidates from these CDRs have been screened for within the three syntenic mouse chromosomes appears to be more exon-based mutations by single-strand conformation polymor- colinear with the human order, suggesting that translocations phism analysis, without success (M. M. Le Beau, personal occurred more frequently than inversions during mammalian evo- communication; refs. 19, 22, and 23). If loss of a single allele were lution. Our comparative map should enhance understanding of the evolution of the del(5q) chromosomal region. Mutant fish harbor- sufficient to contribute to the aberrant development of myeloid ing deletions affecting the 5q CDR syntenic region may provide progenitors (24, 25), one would not expect classical tumor useful animal models for investigating the pathogenesis of my- suppressor gene searches, which ultimately rely on detection of elodysplastic syndrome and acute myeloid leukemia. a mutation in the undeleted allele, to be an optimal method for identifying tumor suppressor genes. The zebrafish forward-genetic model offers several unique onrandom translocations and recurring deletions of specific advantages in defining the role of 5q deletions in MDS͞AML Ngenomic regions are frequently observed in cases of my- pathogenesis. First, hematopoietic genes and signal transduction elodysplastic syndrome (MDS) and acute myeloid leukemia pathways are highly conserved between humans and zebrafish (AML). Although the cloning and characterization of fusion (26–29). Second, both a genome-wide, ␥-irradiation-based genes generated by chromosomal translocations have helped to method of randomly introducing deletions or mutations and clarify the pathogenesis of these hematologic malignancies, the PCR-based identification of genomic fragment-specific dele- contribution of the loss of genes within recurring deletions tions are well established in zebrafish (30). Finally, there is remains an enigma (1). Interstitial loss of all or part of the long considerable evolutionary conservation (synteny) of genomic arm of , del(5q), is one of the most frequent somatically occurring clonal chromosomal deletions in MDS and regions between humans and zebrafish. Some reports estimate AML (1–3). Approximately 42% of patients with therapy- an 83.1% frequency of conserved syntenies among the 804 related MDS͞AML (4–6) and 10–15% of those in whom these orthologous gene pairs shared by humans and zebrafish, com- diseases arise de novo (7, 8) show loss of heterozygosity within pared to 90.4% for 375 mouse–human gene pairs (31–33) and the long arm of chromosome 5. Notably, the deletions within 5q 40–50% for pufferfish–human syntenies (34). are interstitial (4, 5) and occurred early in the development of Here we report the identification and characterization of a hematopoietic stem cells (9–11). zebrafish region on linkage group 14 (LG14) that is syntenic with Many groups have worked diligently to identify and refine the the entire region of human chromosome 5q, containing both the critical deleted regions (CDRs) on human chromosome 5 and ultimately to identify the tumor suppressor genes in these regions This paper was submitted directly (Track II) to the PNAS office. (1–7). Using standard cytogenetic techniques, fluorescence in Abbreviations: MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; CDR, critical situ hybridization and loss-of-heterozygosity analysis based on deleted region; RH, radiation hybrid; LG, linkage group; z5qsCDR, zebrafish 5q syntenic genetic polymorphism, these groups identified two CDRs in CDR; EST, expressed sequence tag. ͞ MDS AML. The proximal CDR within chromosome band 5q31 § ࿝ Ͼ To whom reprint requests should be addressed. E-mail: thomas [email protected]. is found in 90% of cases (2–5, 12–15) and was originally defined The publication costs of this article were defrayed in part by page charge payment. This as the region centromeric to D5S436 and telomeric to IL-9 (4). article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Subsequent studies refined this commonly deleted region to §1734 solely to indicate this fact.

6136–6141 ͉ PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.072560099 Downloaded by guest on September 30, 2021 proximal and distal 5q CDRs most frequently lost in human distributed along the region between IL9 and D5S1955: 70 in the MDS͞AML and 5qϪ syndrome. proximal 5q31 CDR between IL9 and D5S436, with 19 in the smallest 5q31 CDR (between D5S479 and D5S500), and 79 Materials and Methods in the distal 5q32–5q33.3 CDR between ADRB2 and IL12B (Fig. Identification and Physical Mapping of the Human 5q CDR. To con- 1 A and B). Six genes are located in the ‘‘GAP’’ region between struct a physical map of the 5q CDR, we searched the updated the proximal and distal CDRs. Seventy-six of these genes had database of the draft (http:͞͞www.ncbi.nlm. putative zebrafish gene͞EST orthologs, whereas 79 did not (Fig. nih.gov and http:͞͞genome.ucsc.edu). In some instances, the 1). RH mapping localized 33 orthologs within a contiguous physical map was augmented with previously published findings, region of LG14, of which 17 were orthologs of proximal 5q31 which were integrated according to their P1 artificial chromo- CDR genes, 15 were orthologs of distal 5q32–33.3 CDR genes, some (PAC)͞bacterial artificial chromosome͞yeast artificial and 1 was an ortholog of a gene in the gap between the proximal chromosome-based physical positions (5, 12, 14, 18, 35, 36). and distal regions (Fig. 2 Center). All 33 orthologs were encom- passed in a region on LG14 flanked by polymorphic markers Zebrafish Genes Orthologous to Genes in Human 5q CDR. To identify Z8873 and Z1396, which spans Ϸ75 cM (Fig. 2 Left). Fifteen zebrafish–human orthologs, we relied on the ‘‘reciprocal best- additional orthologs were located on LG21 between z5193 and hit’’ method, as described (31, 33). In brief, the sequences z10508, 11 of which are orthologs of CDR genes on distal of all genes within human 5q CDRs were compared to a subset chromosome band 5q32–33.3. Two of the genes are located on of nonredundant zebrafish sequences and the DBEST database LG10 and LG6 (Fig. 1 A and B). (http:͞͞www.ncbi.nlm.nih.gov͞blast͞blast.cgi) by using TBLASTN. The forward search usually generated one of three Comparative Analysis and Identification of Zebrafish Genomic Frag- results: a single zebrafish cDNA [either a characterized gene or ments Syntenic to Human 5q CDRs. Comparative genome mapping an expressed sequence tag (EST) homolog], two related ze- relies on the identification of orthologous gene loci in two brafish cDNAs, or no match with the known zebrafish cDNAs. different species that have descended from a single locus in the Thus, it was necessary to perform 3Ј-untranslated region se- last common ancestral organism. Because the lack of full-length quence alignment with DNASTAR software to exclude the possi- ORFs in the zebrafish ESTs precluded phylogenetic tree anal- bility of a contiguous sequence from identical clones. If all clones ysis, we resorted to a reciprocal best-hit search (31, 33) to identify were unique and noncontiguous, they were considered orthologs putative orthologs. of genes located within the human 5q CDRs. Zebrafish genes Thirty-three zebrafish cDNAs were identified as orthologs of and ESTs showing the highest level of sequence identity (max- human 5q CDR genes and were distributed on LG14 between imum BLAST probability, e-20) were further compared against Z8873 and Z1396. This region spans 54 Mb (roughly correspond- the National Center for Biotechnology Information human ing to 75 cM, based on the 720 kb͞cM distance estimated for the nonredundent protein sequence database by BLASTX reverse sex-averaged map), or approximately twice as long as its human searching. The identification of zebrafish orthologs was con- 5q counterpart fragment (Ϸ27 Mb). We termed this region the firmed if the human genes used in the original search retained zebrafish 5q syntenic CDR (z5qsCDR), which contains at least their positions as the closest match after the BLASTX search. 39 orthologous pairs and is highly syntenic with human 5q31.1– Mouse orthologs were identified as described above or by 33.3, representing a stretch of DNA that has been conserved HOMOLOGGENE or UNIGENE database searches (http:͞͞ since the divergence of human and zebrafish genomes Ϸ450 www.ncbi.nlm.nih.gov͞UniGene). Map positions were obtained million years ago. from the mouse genome database (http:͞͞www.informatics.jax. The order of genes within this large syntenic region appears to org͞or http:͞͞www.ncbi.nlm.nih.gov͞Homology). have been reshuffled during evolution (Fig. 2 Left). In general, the upper section of the syntenic region corresponds more Mapping of Zebrafish Orthologs with a Radiation Hybrid Panel. closely to the human proximal 5q31 CDR, whereas the middle Radiation hybrid (RH) mapping was performed on the Good- and lower parts are more syntenic with the distal 5qϪ syndrome GENETICS fellow zebrafish T51 RH panel, which was developed by fusing CDR (5q32–33.3). This finding is consistent with the disrupted irradiated zebrafish fin AB9 cells to hamster Wg3H cells (37). In gene order reported for zebrafish-human syntenies, such as brief, primer pairs are designed from the 3Ј-untranslated regions LG17-Hsa 14, LG8-Hsa1, and LG3-Hsa17 (32, 33), indicating of putative orthologous gene or EST sequences obtained from that significant intrachromosomal rearrangements and inver- http:͞͞www.ncbi.nlm.nih.gov and http:͞͞zfish.wustl.edu͞. sions have developed in fish and mammalian lineages since the Among ESTs of which only 5Ј sequences were available, we used divergence from their last common ancestor. 3Ј-rapid amplification of cDNA ends to obtain 3Ј sequences. All We also compared syntenic relationships among humans, primer sequences were selected with OLIGO 6 software. Each mice, and zebrafish (Fig. 2 Right). Although the 5q CDR gene or EST was positioned relative to frame markers on the T51 orthologs have remained largely intact in humans and zebrafish, panel by using the SAMAPPER 1.0 RH mapping program. Addi- they are distributed among chromosomes 18, 11, and 13 in mice. tional mapping information can be found at http:͞͞ This finding should not be considered unusual because another 134.174.23.167͞zonrhmapper͞and http:͞͞zfin.org͞. group of genes that has remained intact during the evolution of zebrafish, cats, and humans was fragmented into four chromo- Results somes in the mouse (32). Nonetheless, the order of genes Identification and Mapping of Zebrafish Gene͞EST Orthologs Within residing in the human 5q CDRs and the three syntenic mouse Human Chromosome 5q CDRs. Two human 5q CDRs were studied: chromosomes appears to be colinear, consistent with the notion (i) the proximal chromosome band 5q31 CDR spanning Ϸ12.4 that chromosomal translocations were fixed more frequently Mb between IL9 and D5S436, which was subsequently refined to than inversions during mammalian evolution (32). a region spanning Ϸ1.5 Mb between D5S479 and D5S500 (Fig. 1A) and (ii) the distal 5q32–33.3 CDR spanning Ϸ11.5 Mb Is the Zebrafish LG21, a Duplicated product of the LG14 Genome between ADRB2 and IL12B (Fig. 1B). All human genes were Region, Syntenic with the Human 5q CDR? Previous studies revealed placed in order according to the Genome Working Draft extensive contiguous or interrupted blocks of synteny, but no (http:͞͞www.ncbi.nlm.nih.gov and http:͞͞genome.ucsc.edu͞ one-to-one correspondence, between zebrafish and human ge- goldenPath͞septTracks.html) and previously published data (5, nomes (31–33). Each zebrafish LG shares conserved syntenies 12, 14, 18, 35). Altogether, 155 genes were physically mapped and with an average of 4.5 (2–7) human chromosomes (31), whereas

Liu et al. PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 ͉ 6137 Downloaded by guest on September 30, 2021 Fig. 1. Physical map of the human chromosome 5q CDR and chromo- somal localization of zebrafish orthologs. Two critically deleted human chromosome 5q regions (proximal and distal) are shown in A and B. The proximal AML͞MDS CDR on chromosome bands 5q31 spans a region between IL9 and D5S436 with the proximal smallest CDR between D5S479 and D5S500 (A, red bar and line). The distal 5qϪ syndrome CDR spans chromosome bands 5q32–33.3 between ARBB2 and IL12B (Fig. 1B, gray bar). One hundred fifty-five genes and 10 sequence-tagged site (STS) markers are listed according to the updated human genome draft at http:͞͞www.ncbi.nlm.nih.gov͞and http:͞͞genome.ucsc.edu͞(Aug. 2001 version) and previous publications (5, 12, 14, 18, 35, 36). Zebrafish or- thologs are listed by clone name for ESTs or accession no. for genes on the right side of the figure. n.a., No available orthologs are found in the current public zebrafish database. The RH mapping positions for 55 zebrafish orthologs are indicated, among which 33 orthologs mapped to LG14, 15 to LG21, 2 to LG10, 2 to LG6, and 1 to LG12. We were unable to map 23 orthologs for technical reasons.

each mammalian chromosome shares syntenies with more than brafish (26–29, 31–33). As many as 83% of the orthologous gene one zebrafish LG (33). Consistent with these observations, the pairs are conserved within syntenic chromosomal regions (33). human 5q CDR chromosome fragment contains at least two The value of a zebrafish model of tumor suppressor genes blocks of synteny with zebrafish LG14 (39 genes or ESTs) and deleted in human MDS͞AML will depend in part on the level LG21 (15 ESTs). In addition, five zebrafish ESTs (fb52g05, of synteny between human chromosome 5q and previously fd19g04, fc15e05, fb15a06, and fc66d05) were unexpectedly unidentified genomic regions in the fish. located on LG10, LG12, and LG6. Of 155 physically positioned genes or protein-coding se- It is worth noting that zebrafish orthologs on LG21 are quences located on human 5q31–33.3 within the proximal and dispersed within the region containing the LG14 orthologs (Fig. distal CDRs, we identified 76 genes͞ESTs as zebrafish orthologs. 1B). Thus, fragmentation of the human 5q CDR into two RH mapping showed that 33 map to zebrafish LG14 and cluster zebrafish chromosomes (LG14 and LG21) seems unlikely. A in the region between Z8873 and Z1396, which we refer to as the more likely possibility is that LG21 and LG14 are duplicates of z5qsCDR. There also are six additional zebrafish genes͞ESTs in each other, both being orthologous to the 5q CDR. In support this region, raising the total to 39 (Fig. 2 Left). We expect to be of this hypothesis, we found two zebrafish orthologs of three able to identify zebrafish orthologs for most of the remaining 79 single-copy human genes on 5q CDR: MSX2 (30), CNOT8, and human 5q CDR genes, as additional zebrafish ESTs and genes TAF2S. One copy of these genes (Msxa, fd59c07 and fj40a05) are sequenced. It also will be interesting to determine whether mapped to LG14 and the other (Msxd, fd17b08 and fc41g11) to LG21 (Fig. 3 Left), indicating that portions of LG21 and LG14 any of these orthologs map to the conserved region on LG14. are duplicates of each other. This new LG14–LG21 duplicated The orders of loci within the chromosome segments of this chromosome segment, combined with 13 previously identified block of conserved synteny are quite different between zebrafish duplicated segments (33), strongly suggests that genome-wide and humans. This fact, together with previous work, suggests that duplication events occurred and were retained during ray-fin chromosomal inversions are fixed in fish and human lineages evolution. more often than translocations. By contrast, mouse genomic regions syntenic with the human 5q CDR can be found on Discussion chromosomes 18, 11, and 13, but the order of genes is better The zebrafish affords a potentially useful model for identifying preserved than in fish. Thus, since their divergence from humans human tumor suppressor genes relevant to MDS͞AML. It offers Ϸ112 million years ago, mice appear to have undergone more the advantage of ‘‘forward genetics’’ (39, 40), and there is rapid genomic evolution than zebrafish, which diverged Ϸ450 considerable conservation of genes, signaling pathways, and million years ago. This may reflect an increase in chromosomal chromosome fragments (syntenies) between humans and ze- translocations during murine evolution.

6138 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.072560099 Liu et al. Downloaded by guest on September 30, 2021 Fig. 2. Chromosomal regions in zebrafish and mice syntenic with the proximal and distal human chromosome 5q CDRs. (Left) Thirty-nine zebrafish orthologs of genes within the human chromosome 5q CDR on LG14 are listed according to their RH mapping position between Z8873 and Z1396 (z5qsCDR), where z6847 is a centromere-linked marker. *, Genes with a second duplicated zebrafish gene on LG21 (see Fig. 3). (Center) Human chromosome proximal 5q CDR region (red),

distal 5q CDR (gray), and their flanking regions (green). (Right) Mouse syntenic regions and the chromosome positions of murine orthologs in three separate GENETICS chromosomal regions are shown on the right.

As vertebrates evolved from their early deuterostome ances- fragmentation or translocation seems an unlikely explanation. tor, the entire genome was duplicated through two rounds of Further study indicated that of 39 genes on z5qsCDR, three are duplication (one-to-two-to-four rule) (41). Accumulating evi- duplicated (fd59c07–fd17b08, fj40a05–fc41g11, Msxa–Msxd) and dence suggests further that the fish genome was duplicated a all map to LG14 and LG21, as the regional duplication model third time to produce up to eight copies of the original deuter- would predict. Thus, the three duplicated gene pairs provide ostome (one-to-two-to-four-to-eight rule) (42, 43). The third critical evidence that portions of LG21 and LG14 are duplicates duplication took place after the divergence of two major radi- of each other and increase the total of zebrafish–human dupli- ations of jawed vertebrates, consisting of ray-finned fish͞ cated gene pairs to 62 (59 were identified previously) (33). This actinopterygia (which includes zebrafish) and the sarcopterygian finding, and the observation that 21 of 25 zebrafish LGs contain lineage (which includes lungfish and all land vertebrates). The duplicate segments (33), strongly suggests that the third dupli- most convincing example comes from research on Hox gene cation event Ϸ450 million years ago could have involved the clusters. Tetrapods possess more than 42 Hox genes arranged in entire genome. four clusters (A–B–C–D), whereas zebrafish have at least 47 As noted earlier, duplicates for 36 of the 39 orthologs on LG14 genes arranged in seven clusters (AaAb–BaBb–CaCb–Da), sug- have not yet been identified. This could reflect missing dupli- gesting a genome duplication in zebrafish followed by the loss of cates due to the redundancy of gene function and relaxed individual genes and clusters (41, 43). selection pressure, the presence of pseudogenes or silencing by We found that human 5q CDRs share syntenies with two mutation (44, 45). However, one could predict that fewer than zebrafish LGs, LG14 and LG21. One interpretation is that LG14 92% of the duplicated genes were lost after the duplication and LG21 are derived from fragmentation of the human 5q CDR event, consistent with current estimates that fewer than 80% of orthologs in ray-finned fish. However, because LG21 contains 15 duplicates are missing after the third duplication (33). Once zebrafish EST orthologs, and human orthologs of both LG14 and sequencing of the zebrafish genome is completed, direct align- LG21 are distributed across essentially the same loci, simple ment of chromosome sequences, as applied to the Arabidopsis

Liu et al. PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 ͉ 6139 Downloaded by guest on September 30, 2021 Fig. 3. Duplication in zebrafish of the human chromosome 5qsCDR. Eighteen ESTs that are orthologous to human chromosome 5q CDR genes are located on LG21 (genetic distance indicated in cM). Three LG21 zebrafish genes (fd17b08, fc41g11, and Msxd; human orthologs, CNOT8, TAF2S, and MSX2) are duplicates of genes located on LG14. Human chromosome 5qϪ syndrome CDR (distal CDR) and the mouse chromosome location of corresponding orthologs are also shown.

thaliana genome (46), will offer the best strategy for addressing Finally, our syntenic map could be used in genomic ‘‘ping- the questions raised here. ponging’’ between the human 5q CDR and z5qsCDR, allowing Taken together, our findings suggest that the zebrafish contain positional cloning of many genes that are mapped to this region. highly evolutionarily preserved chromosome fragments ortholo- gous to the human 5q CDR chromosome fragment. Zebrafish We thank Anne Delahaye-Brown and AnHua Song for technical assis- mutants harboring deletions that disrupt the z5qsCDR should tance, John Gilbert for editorial review, and Doris Dodson for assistance provide a valuable model to investigate the molecular patho- with manuscript preparation. This work was supported by a National genesis of AML͞MDS, especially, in view of the conservation of Institutes of Health Grant CA 93152 and by a Leukemia and Lymphoma myeloid cell development between zebrafish and man (47). Society Center grant.

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