GENOMICS 4, 206-209 (1989)

SHORT COMMUNICATION Chromosomal Localization of the Human Homeo

Box-Containing Genes, EN7 and E/V2

CAIRINE LOGAN,* HUNTINGTON F. W~uAm,t JOHANNA M. ROMMENS,* AND ALEXANDRA L. JOYNER”

‘Divison of Molecular and Developmental Biology, Mount Sinai Hospital Research Institute, and Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada; tDepartment of Medical Genetics, University of Toronto,

Toronto, Ontario, Canada; and SDepartment of Genetics, Hospital for Sick Children, Toronto, Ontario, Canada

Received July 21, 1988; revised September 30, 1988

The human homologs of the mouse homeo box-con- taining genes, En-1 and En-2, which show homology to the Drosophila engrailed gene, have been isolated. The human EN1 gene was mapped to chromosome 2 by analysis of mouse-human somatic cell hybrids. The human EN2 gene was localized to chromosome 7, 7q32-7qter, by analysis of rodent-human somatic cell hybrids and cell lines carrying portions of chromosome 7. Cc 1989 Academic Press. Inc.

A number of potential developmental genes in ver- tebrates have been identified (e.g., Levine et al., 1984; McGinnis et al., 1984) on the basis of their sequence homology to the homeo box domain of many Drosophila pattern formation genes (reviewed by Gehring, 1987). We have been studying the mouse homeo box-contain- ing genes, En-l and En-2 (Joyner et al., 1985a; Joyner and Martin, 1987), which were identified because of their extensive sequence homology with the Drosophila segmentation gene engrailed (Poole et al, 1985). Our recent in situ RNA hybridization analysis of the expression of these genes during mouse embryogenesis suggests that they may play a basic role in brain de- velopment. Expression of both genes begins soon after formation of the neural ectoderm in essentially iden- tical bands of cells across the neural folds (Davis et al., 1988; C. Davis and A. Joyner, 1988). Expression con- tinues in a similar region of the neural tube throughout development and in overlapping but different sets of cells in the adult cerebellum and pons.

As a first step toward identifying and studying the human engrailed-like genes, we have isolated a number of overlapping human genomic clones using mouse En-l and En-2 cDNA clones as probes. A detailed mo- lecular analysis of the structure, sequence, and expres- sion of these human clones, which we designate EN1 and EN2 (C. Logan and A. Joyner, manuscript in preparation), has confirmed that they represent the

human homologs of the mouse En genes. Here we re- port the chromosomal localization of each gene.

Figure 1 shows restriction maps of the EN1 and EN2 genomic regions represented in the overlapping phage clones isolated. The filled boxes mark the location of the respective homeo boxes. The fragments labeled mp3 and mp4 identify two single-copy “mapping probes” used for these studies. On Southern blots of EcoRI- digested human genomic DNA, mp3 detects an 18-kb EN1 and mp4 detects a 3.6-kb EN2 restriction frag- ment (see Fig. 2). In addition, mp3 hybridizes to a 6.8- kb mouse En-l EcoRI genomic fragment due to cross- homology between the genes (C. Logan and A. Joyner, unpublished data).

To determine the chromosomal localization of each human gene, the two mapping probes were used to screen Southern blots of EcoRI-digested DNA from 20 mouse-human hybrid cell lines retaining various com- plements of human chromosomes (Willard et al., 1985; Tsui et al., 1986). An example of these results is shown in Fig. 2, and Table 1 summarizes all of the data ob- tained. The human genes EN1 and EN2 can be un- ambiguously assigned to chromosomes 2 and 7, re- spectively. Furthermore, one of the cell lines, t60-14 (Tsui et al., 1986), which was negative with the mp4 probe, contains a deleted chromosome 7 (7pter-7q31:), suggesting localization of EN2 to the distal long arm. To confirm this result and to further localize EN2, a second panel of 12 rodent-human hy- brids and patient cell lines containing various portions of human chromosome 7 (Zengerling et al., 1987) were screened with the mp4 probe. As shown in Table 2, only those lines containing the distal tip of the long arm of chromosome 7 were positive. The shortest frag- ment of chromosome 7 contained in one of these lines included 7q32-qter and hybridized to the mp4 probe. Taken together, these data position EN2 on chromo- some 7 between 7q32 and 7qter.

In the mouse, mapping studies of the homeo box- containing genes have helped to elucidate both the

0888.7543/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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5’

5’

S BS s

I I/ I

mp3 -

S

In .

3’ S E s SEI E

I I I \I I

EN1

mm -

HE EHB B HE E B E H E E

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lx? 1 kb

FIG. 1. Restriction maps of human EN1 and EN2. Each horizontal line represents a composite of the genomic fragments contained in the overlapping phage clones isolated for each gene. The filled boxes represent the homeo boxes. The “mapping probes” mp3 and mp4 are indicated by open boxes. mp3, a 1.6-kb HindlII/SstI fragment, and mp4, a 1.7-kb Sat1 fragment, were subcloned into pucllb. S, &?I; B, BarnHI; E, EcoRI; H, HindIII.

chromosomal arrangement of these genes and their possible allelic relationships with known developmen- tal mutations. More than 14 mouse genes containing homeo boxes homologous to the Drosophila antenna- pedia homeo box (“HOC genes; Martin et al., 1987) have been isolated and found to reside in clusters on four different chromosomes. The mouse genes En-l and En-2, which have extensive homology to the closely linked Drosophila genes engrailed and invected, reside on two other mouse chromosomes, 1 and 5, respectively.

12345MH

-18kbH ENI

- 6.8 kb M m

s- *

- 3.6 kb H &E

FIG. 2. Southern blot hybridization of mouse-human hybrid cell lines. Southern blot analysis was carried out as previously de- scribed 110). Six micrograms of mouse CM), human (H), and hybrid cell line (l-6) DNAs were digested with EcoRI, separated by elec- trophoresis on a 0.7% agarose gel, transferred to GeneScreen, and crosslinked by exposing the filters to uv light. DNA fragments from the plasmids mp3 and mp4 were isolated in low-melt agarose and random-primed to a specific activity of 2 X 10’ cpm pg-’ (8). The blot was hybridized and washed under stringent conditions. Hybrids 1, 2, and 6 contain human chromosome 7, hybrid 3 contains both human chromosomes 2 and 7, and hybrid 4 contains human chro-

Recombinant inbred and recombinational analyses (Joyner and Martin, 1987; Martin and Joyner, manu- script in preparation) have shown that En-l maps within 0.5 CM of, but is not allelic with, Dominant hemimelia (Green, 1981) and En-2 maps within 1.5 CM

TABLE 1

Summary of the Southern Blot Analyses of 20 Mouse-Human Hybrid Cell Line DNAs Probed

with the EN1 (mp3) and EN2 (mp4) Probes

Discordant fraction

Chromosome ENI (mp3) EN2 (mp4)

1 9/20 9/20 2 o/20 12/20 3 9/20 10/20 4 7/20 10/20 5 6/20 B/20 6 7/20 11/20 7 1 l/19 O/18 8 9/20 7/20 9 10/20 12/20

10 13/20 7/20 11 B/19 11/19 12 lo/no 8/20 13 B/20 12/20 14 11/20 7/20 15 9/20 14/20 16 10/20 10/20 17 3/20 11/19 18 B/20 12/20 19 6/20 B/20 20 9/20 7/20 21 7/20 7/20 22 9/20 13/20 X B/18 7117 Y 11/20 11po

Note. The fraction of discordant hybrids is listed in each column. Denominators less than 20 reflect hybrids not scored because they contained chromosome rearrangements or translocations involving

mosome 2. chromosomes 7, 11, 17, and X.

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TABLE 2

Summary of the Southern Blot Analyses of 13 Rodent-Human Hybrid Cell Line and Patient

Cell Line DNAs Carrying Various Portions of Human Chromosome 7 Probed with

the EN2 (mp4) Fragment

Cell line Region EN2 (mp4)

5387 3 cl 10 Whole 7 only + Ru-Rag 6-19 de17 (qter-ten:) + Ru-Rag 4-13 de17 (pter-ten:) -

Ru-Ragla5-11 de17 (qter-ten:) + MH-Rag 8-7 t(7;15)(7qter-q32::15ql3-qter) + IT-9 2-21-14 t(6;7)(6qter-q12::7pl4-qter) + 194 Rag 6-13 de17 (pter-q32:), 3q- 1059 Rag 5 de17 (pter-q22::q32-qter) + 2068 Rag 22-2 t(6;7)(6pter-q27::7q22-qter) + 4AF1/106/K015 7 only + lCF2/5/K016 7pll-q22 -

lEF2/3/K017 (7pll-q22)(7q22-qter) + t60-14 (7pter-q31:)

Note. The amount of human chromosome 7 material retained in each cell line is indicated. 1059 Rag 5 and 2068 Rag 22-2 (Jobs et al., manuscript in preparation); 4AF1/106/K015,1CF2/5/K016, and lEF2/3/K017 (Arfin et al.. 1983; Zengerling et al., manuscript in preparation); t60-14 (Tsui et al., 1986); other hybrids (Zengerling et al., 1987).

of, but is not allelic with, Hemimelic extra toes (Green, 1981).

Human homologs of many of the mouse Hex genes have been identified and found also to reside in clusters on four chromosomes (e.g., Bucan et al., 1986; Canniz- zaro et al., 1987; Joyner et al., 1985b; Rabin et al., 1986). Our present studies show that the human EN1 and EN2 genes are not linked to each other and map to chromosomes 2 and 7, respectively. Unlike the mouse, human HOX5 (HHOcl3) and HOXl map to the same chromosomes as the EN genes (Bucan et al., 1986; Cannizzaro et al., 1987; Rabin et al., 1986). However, HOXl, which maps to human chromosome 7, resides on the short arm (Bucan et al., 1986; Rabin et al., 1986), whereas EN2 maps to the distal tip of the long arm.

In the mouse, En-2 lies proximal to a conserved linkage group that includes the genes Pep-7, Pgm-1, and Afp (Nadeau and Reiner, 1988) but which is ho- mologous with human chromosome 4q. There is, how- ever, a second more distal linkage group that includes the genes Gus and Mor-1 and shows homology with human chromosome 7. In the human, GUSB maps to 7q and part of a linkage group showing homology to mouse 6 maps distal to GUSB (Tsui, 1988). EN2 may therefore map distal to or interrupt this linkage group for which TCRB is presently the distal-most gene. On mouse chromosome 1, two linkage groups surround En-l (Nadeau and Reiner, 1988). En-l maps within 5 CM of the distal group which extends proximal to at least Cfh (Seldin et al., 1988); however, this group is

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homologous to genes on human chromosome lq. Per- haps of significance, the proximal linkage group, which includes Idh-I and Cryg-1, is homologous to genes on human chromosome 2q33-37. If En-l is part of this linkage group, then it would mark a new distal bound- ary. However, Hox-5, which lies on mouse chromosome 2 (Featherstone et al., 1988), also maps to this same region of human chromosome 2 (2q31-37; Cannizzaro et al., 1987), suggesting that EN1 and HOX5 may be linked in human. This would present a chromosomal arrangement of these homeo box-containing genes in human different from that in mouse.

Although no human developmental mutations have been assigned to the distal tip region of chromosome 7 containing EN2, many developmental abnormalities, including microcephaly, have been associated with ter- minal 7q deletions (reviewed in Tsui, 1988) and more precise localization of EN1 is required. In our prelim- inary experiments we have identified three restriction length polymorphisms for EN2. Thus, we expect that the EN genes will serve as useful markers for future mapping studies and may ultimately be implicated in genetic lesions affecting brain development.

ACKNOWLEDGMENTS

Thanks to Lap-Chee Tsui for providing a panel of rodent-human somatic hybrid cell lines and patient cell lines containing whole or portions of human chromosome 7. This work was supported by grants from the Medical Research Council (MRC) of Canada (A.J. and H.F.W.). C.L. was supported by an MRC Studentship and J.M.R. was supported by an MRC Fellowship. A.J. is an MRC Scholar and H.F.W. is an MRC Scientist.

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