The murine polycomb-group genes Ezh1 and Ezh2 map close to Hox
gene clusters on mouse Chromosomes 11 and 6
** Andrew R. Haynes,
** Angelika Lebersorger,
Steve D.M. Brown,
Research Institute of Molecular Pathology (IMP) @ The Vienna Biocenter, Dr. Bohrgasse 7, A-1030 Vienna, Austria
Medical Research Council UK Mouse Genome Centre, Harwell, Oxfordshire, OX11 ORD, UK
INSERM U491, Ge´ne´tique Me´dicale et De´veloppement, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France
Received: 29 September 1998 / Accepted: 17 November 1998
Polycomb-group (Pc-G) genes encode chromosomal proteins that
restrict expression patterns of the homeotic selector genes (HOM-
C cluster) and presumably many other developmentally important
loci (reviewed by Paro and Harte 1996). Of the ≈40 Pc-G loci that
have been predicted from genetic analyses in Drosophila (Ju¨rgens
1985), currently 11 Pc-G genes have been isolated (reviewed by
Pirrotta 1997). Among these, Enhancer of zeste [E(z)] (Jones and
Gelbart 1993) seems to provide a central role(s), because it is one
of only two Pc-G genes that appear to be conserved in lower
eukaryotes (Korf et al. 1998). In addition to homeotic transforma-
tions, loss-of-function mutations of Drosophila E(z) also result in
maternal sterility and reduced mitotic indices during early embryo-
genesis (Phillips and Shearn 1990). E(z) mutants can induce partial
decondensation of polytene chromosomes (Rastelli et al. 1993), a
phenotype that is further reflected by increased chromosome
breakage and cell cycle defects (Gatti and Baker 1989). Thus, E(z)
appears to be a pleiotropic Pc-G gene with functions in chromatin
architecture, gene regulation, and growth control.
A crucial role(s) for Pc-G activity during normal and perturbed
development has been corroborated by the functional analysis of
several mammalian Pc-G homologs, some of which are implicated
in the onset of leukemia (reviewed by van Lohuizen 1998). Al-
though loss-of-function mutations of mammalian E(z)-related
genes are currently not described, a human E(Z) homolog has been
shown to associate with the VAV proto-oncoprotein (Hobert et al.
1996b), an interaction that suggests involvement in signal-
dependent T-cell proliferation. In addition, E(z)-related genes
share the SET domain (Tschiersch et al. 1994)—which is charac-
teristic for the SET family of chromatin regulators (Jenuwein et al.
1998)—with the human trithorax homolog ALL-1/HRX, another
proto-oncogene that is frequently disrupted by 11q23 transloca-
tions in acute leukemias (Tkachuk et al. 1992; Djabali et al. 1992).
Since the SET domain appears to be a target for phosphorylation-
dependent signaling pathways (Cui et al. 1998), mammalian E(z)-
related genes are likely to participate in developmental switches
that may trigger differentiation or proliferation.
We have recently isolated murine (Ezh1) and human (EZH2)
cDNAs of mammalian E(z) homologs (Laible et al. 1997). To
characterize the genomic organization and chromosomal assign-
ment of the two murine Ezh loci, we already identified a genomic
Ezh1 clone from a cosmid C57B1/6 library (Laible et al. 1997).
Sequence analysis over a length of ≈28 kb and comparison with the
full-length Ezh1 cDNA indicated that the complete Ezh1 coding
information is separated into 20 exons (Fig. 1, top). Using a PCR-
amplified DNA probe that encodes amino acids 335–430 of Ezh2
(within this region, Ezh1 and Ezh2 cDNAs display only 51% iden-
tity; Laible et al. 1997), we have now isolated a ≈15 kb, partial
genomic Ezh2 subclone that, in analogy to genomic Ezh1, com-
prises exons 4 to 12 (Fig. 1, bottom). Reduced stringency hybrid-
izations of digested mouse DNA with specific Ezh1 or Ezh2 DNA
probes exclusively reveal fragments that can be correlated with
predicted sizes from either the genomic Ezh1 or Ezh2 clones, thus
confirming the presence of only two Ezh loci in the mouse genome
(Laible et al. 1997 and data not shown). To generate DNA probes
for subsequent examination of chromosomal localization by fluo-
rescence in situ hybridisation (FISH) we subcloned the indicated
12 kb Ezh1 and8kbEzh2 genomic fragments into pBluescript
FISH analyses were performed on metaphase spreads prepared
from Concanavalin A-stimulated lymphocytes that had been de-
rived from WMP male mice, which are homozygous for metacen-
tric Robertsonian (Rb) translocations in all autosomes except Chr
19. The genomic Ezh1 and Ezh2 DNA probes were biotinylated by
nick translation with biotin-16-dUTP, preincubated with a 150-
fold excess of murine Cot-1 DNA (Gibco-BRL), and hybridized to
the metaphase spreads as described (Pinkel et al. 1986). Biotin-
labeled DNA was detected by fluorescein isothiocyanate (FITC)-
conjugated avidin (Vector Laboratories). Chromosomes were
counterstained and R-banded with propidium iodide. For each of
the two genomic Ezh probes, 50 metaphase spreads were analyzed,
indicating specific signals in ≈80% of the preparations. The Ezh1
probe revealed a symmetrical, two-dotted staining of the C-D re-
gion of mouse Chr 11 present in the Rb (1;11) translocation (Fig.
2, left panel), whereas the Ezh2 probe specifically hybridized with
the B region of mouse Chr 6 present in the Rb (4;6) translocation
(Fig. 2, right panel).
The chromosomal position of the two murine Ezh loci was
further defined by haplotype analysis of recombinant progeny
from the European collaborative interspecific backcross (EUCIB;
Breen et al. 1994; Rhodes et al. 1998). The EUCIB backcross is
derived from matings between M. spretus and C57BL/6. Back-
cross progeny were produced by mating (M. spretus × C57BL/6)
females to either M. spretus or C57BL/6 mice. Parental M.
spretus and C57/B16 allelic variants were identified on a series
(three each for Ezh1 and Ezh2) of PCR-amplified DNA fragments
by single-stranded conformation polymorphism (SSCP). Reaction
products were electrophoresed on 8% non-denaturing polyacryl-
amide gels, with migration differences being revealed by a silver
* Present address: Dairy Science Group, AgResearch, Private Bag 3123,
Hamilton, New Zealand.
** These authors contributed equally to this work.
Correspondence to: T. Jenuwein
Accession numbers. The genomic Ezh1 (accession number AF104360) and
genomic Ezh2 (accession number AF104359) sequences have been depos-
ited in GenBank. The fine mapping data of the murine Ezh1 and Ezh2 loci
presented in this study have been submitted to MGD and can be accessed
under accession number J:50304.
Mammalian Genome 10, 311–314 (1999).
© Springer-Verlag New York Inc. 1999