The Murine Polycomb-Group Gene eed and Its Human Orthologue: Functional Implications of Evolutionary Conservation Armin Schumacher, 1 Olivier Lichtarge,* Stuart Schwartz, and Terry Magnuson 2 Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106-4955; and * Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030 Received June 15, 1998; accepted July 31, 1998 Similar to Drosophila, murine Polycomb-group (PcG) genes regulate anterior–posterior patterning of segmented axial structures by transcriptional re- pression of homeotic gene expression. The murine PcG gene eed (embryonic ectoderm development) en- codes a 441-amino-acid protein with five WD motifs which, except for the amino terminus, is highly ho- mologous to Drosophila ESC (Extra Sex Combs). Here, sequence and expression analysis as well as chromosomal mapping of the human orthologue of eed is described. Absolute conservation of the hu- man eed protein along with significant divergence at the nucleotide level reveals functional constraints operating on all residues. The human orthologue appears to be ubiquitously expressed and maps to chromsome 11q14.2– q22.3. Using the first WD motif of the -subunit of the bovine G protein as a struc- tural reference, the predicted locations of two pre- viously identified eed point mutations (A. Schuma- cher et al., 1996, Nature 383: 250 –253) are also reported herein. The proline substitution (L196P) in the second WD motif of the l7Rn5 3354SB null allele maps to the internal core of the inner end of the -propeller blade and is likely to disrupt protein folding. In contrast, the asparagine substitution (I193N) in the second WD motif of the hypomorphic l7Rn5 1989SB allele maps onto the surface of the -pro- peller blade near the central cavity and may affect surface interactions without compromising propel- ler packing. These results illustrate the critical im- portance of all residues for eed function in mammals and support a model whereby the amino terminus might implement function(s) related to embryonic development in higher organisms. © 1998 Academic Press INTRODUCTION In the mouse, a considerable number of developmen- tal control genes have been identified by mutational analysis (Reith and Bernstein, 1991; Copp, 1995; Meisler, 1996). One of these genes is encoded by the classical mouse gastrulation locus eed (embryonic ecto- derm development), which disrupts anterior–posterior (A-P) patterning of the early primitive streak (Niswan- der et al., 1988, 1989; Faust et al., 1994; Holdener et al., 1995a). eed was isolated recently by positional cloning and encodes a 441-amino-acid protein with five WD motifs (Schumacher et al., 1996). The protein is 55% identical and 74% similar to Drosophila ESC (Extra Sex Combs) (Gutjahr et al., 1995; Sathe and Harte, 1995a; Simon et al., 1995), and this high degree of evolutionary conservation comprises 83% of the Eed sequence without a single gap or insertion (Schuma- cher et al., 1996). Remarkably, the amino terminus (residues 1–76) has evolved at a much faster rate than the large WD-motif-containing region and conserva- tion is as low as 24% identity and 41% similarity. esc is a member of the large Polycomb-group (PcG) of genes and as such is required for maintenance of tran- scriptional repression of homeotic genes (Struhl, 1981; Struhl and Akam, 1985; Struhl and White, 1985; Gut- jahr et al., 1995; Sathe and Harte, 1995a,b; Simon et al., 1995). Evidence is increasing that PcG function is executed by multimeric protein complexes modifying higher order chromatin structures, thereby repressing transcription of homeotic and other putative target genes (Paro and Harte, 1996). Accordingly, loss-of- function mutations in Drosophila PcG genes cause de- repression of homeotic genes resulting in transforma- tion of all body segments to an eighth abdominal segment identity (Simon, 1995). Functional analysis of murine homologues of Drosophila PcG genes indicates conservation of transcriptional regulation of homeotic genes across species (Schumacher and Magnuson, 1997; Gould, 1997; van Lohuizen, 1998). For example, conservation of eed function was evident by the finding of highly penetrant, dosage-sensitive posterior ho- 1 Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. 2 To whom correspondence should be addressed at the Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, BRB 609B, Cleveland, OH 44106-4955. Telephone: (216) 368-2254. Fax: (216) 368-3432. E-mail: trm4@po.cwru.edu. GENOMICS 54, 79 – 88 (1998) ARTICLE NO. GE985509 79 0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.