Mammalian Genome 2: 72-75, 1992

9 Springer-VerlagNewYorkInc. 1992

Mapping of a mouse homolog of a Heterochromatin protein gene to the X Chromosome Renata M.J. Hamvas, ~ Wolf Reik, 2 Stephen J. Gaunt, 2 Stephen D.M. Brown, 1 and Prim B. Singh 2 1Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, London W2 IPG, UK; ZDepartment of Molecular Embryology, Institute of Animal Physiology and Genetics Research, Cambridge Research Station, Babraham, Cambridge CB2 4AT, UK Received May 28, 1991; accepted June 12, 1991

Abstract. Modifiers of position-effect-variegation in Drosophila are thought to encode proteins that are either structural components of heterochromatin or enzymes that modify these components. We have recently shown that a sequence motif found in one Drosophila modifier gene, Heterochromatin protein 1 (HPI), is conserved in a wide variety of animal and plant species (Singh et al. 1991). Using this motif, termed chromo box, we have cloned a mouse candidate modifier gene, M31, that also shows considerable sequence homology to Drosophila HP1. Here we report evidence of at least four independently segregating loci in the mouse homologous to the M31 cDNA. One of these loci--Cbx-rsl--maps to the X Chromosome (Chr), 1 cM proximal to Amg and outside the X-inactivation center region.

Chromosomal rearrangements that place a gene next to heterochromatin sometimes result in mosaic or variegated expression of the gene (Spofford 1976). Such variegated position effects are thought to be due to heritable repression, in some cells, of genes close to the breakpoint by spreading of the heterochromatic confirmation into the euchromatin. In Drosophila, the degree of variegation can either be suppressed or enhanced by several dominant mutations, known as modifiers of variegation (reviewed in Eissenberg 1989). Cloning and characterization of one of these modifier genes, HP1, has shown it to be allelic to Suvar(2)5 (Eissenberg et al. 1990) and a structural component of heterochromatin (James and Elgin 1986). Interestingly, sequencing of the Drosophila Polycomb (Pc) gene, a repressor of homeotic genes (Gaunt and Singh 1990; Paro 1990), has revealed a region of homology with HP1 (Paro and Hogness 1991). We Offprint requests to." S.D.M. Brown

have used a PCR-generated probe encompassing this region, termed chromo box, to screen an 8.5 day old mouse embryo cDNA library (Singh et al. 1991). Several different clones were isolated and one large cDNA of the M31 class was sequenced--hereafter referred to as M3t. A stretch of 37 amino acids was uncovered that shares 70% homology with the chromo box domain of HP1. Overall, M31 shows 51% homology at the amino acid level with HP1. M31 was found to be expressed in all adult tissues and in all embryonic tissues, paralleling the ubiquitous expression of HP1 in Drosophila embryos. The chromo box motif appears to be highly conserved in both the animal and plant kingdoms; positive signals are seen in DNAs from a wide range of species probed with HPI chromo box (Singh et al. 1991). Given the apparent role of HPI in heterochromatin and gene repression in Drosophila, we sought to explore the possible role of its murine homologs in the processes of mammalian X Chr inactivation. X Chr inactivation in mammals is thought to occur in three stages (Brown 1991; Lyon 1991). Initiation of X-inactivation is thought to begin at the X-inactivation center (Rastan and Brown 1990). This signal then spreads in both directions inactivating the chromosome. Finally, once inactivated, the X Chr is maintained in this state in successive cell generations through the lifetime of the organism, except in germ cells, where reactivation takes place. In this report, we have identified an X-linked locus with homology to M31, but it maps outside the X-inactivation region, and is therefore unlikely to play a primary role in the initiation of X Chr inactivation. In order to identify and map chromo box loci on the mouse X Chr, we have utilized an interspecific Mus domesticus/Mus spretus backcross segregating the mouse mdx mutation (Cavanna et al. 1988; Keer et al. 1990). The backcross involved the mating of female C57BL/10 mdx/mdx lab mice to male M. spretus. Fe-

R.M.J. Hamvas et al.: Mapping mouse homolog to Chr X

male F 1 progeny were backcrossed to male C57BL/10 mdx/mdx mice. DNAs obtained from the progeny of this cross have been used for mapping both on the mouse X Chr (Brockdorff et al. 1991; Cavanna et al. 1988; Keer et al. 1990), and on mouse Chr 16 (Irving et al. 1991). Hybridization of the M31 cDNA probe (Singh et al. 1991) to Taq I digests of M. spretus detected four major bands of 6.0, 4.0, 2.1 and 1.4 kb (labeled S1-$4 respectively; Fig. 1), and four major bands of 7.0, 4.5, 2.5 and 2.0 kb in C57BL/10 (labeled C1-C4 respectively; Fig. 1).

Fig. 1. Four independent loci show homology to the M31 probe. DNAs from the mdx interspecific backcross were digested with Taq I, size-fractionated on 0.8% agarose gels and blotted onto Hybond-N membranes (Amersham). Hybridization was carried out for 16 h at 65~ in 1% SDS, 6 x SSC, 10% dextran sulphate, 10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, and 1 x Denhardt's solution and 10 ~g/ml denatured sheared salmon sperm DNA. 1 x 10 6 cpm of labeled M31 cDNA probe was used per ml of hybridization mix. Filters were washed in 1 x SSC, 0.1% SDS at 65~ for 90 min before being exposed to autoradiographic film for seven days with intensification. The M31 cDNA is 1.4 kb in length and the entire 1.4 kb was used as probe. C57BL/10 (C) DNA, M. spretus DNA (S) and five progeny DNAs (13, 6, 21, 17 and 20) are illustrated. Mouse 20 is female, the remainder male. Each of four M. spretus alleles (S 1-$4) of 6.0, 4.0, 2.1 and 1.4 kb and four C57BL/10 alleles (C1-4) of 7.0, 4.5, 2.5 and 2.0 kb are indicated. The presence (+) or absence ( - ) of each allele in the five backcross progeny is indicated. In addition, mice 21, 17 and 20, probed with M31 from a longer gel run are shown to illustrate better the clear separation of the $3 and C4 alleles and the segregation of the $3 allele independent of other S alleles; this has entailed running the $4 allele off the gel bottom.

73

The segregation pattern of these bands was studied in progeny derived from the mdx cross. All four M. spretus bands segregated independently of each other, indicating the presence of four loci (see details in Fig. 1). However, the 4.5 kb (C2) C57BL/10 band gave an X-linked pattern of inheritance, absent in some male mice (see mouse 21, Fig. 1). In those male mice where the 4.5 kb (C2) C57BL/10 band was absent, the 4.0 kb ($2) M. spretus band was uniformly present. Thus, the 4.0 kb ($2) M. spretus band is allelic to the X-linked 4.5 kb (C2) C57BL/10 band. Of 38 males scored for M3! and a number of other X Chr markers, 22 had only the M. spretus M31 4.0 kb ($2) allele, 16 had the C57BL/10 4.5 kb (C2) allele and none had both alleles. Of the 38 females scored, 13 had only the C57BL/10 allele and 25 were heterozygous. These results demonstrate that the 4.0 kb and 4.5 kb bands detected by the M31 cDNA probe are X-linked alleles at a locus called Chromo box related sequence I (Cbx-rsl). None of the remaining three C57BL/10 bands segregated amongst the backcross progeny, indicating the presence of three further autosomal loci homologous to M31 in the mouse genome, in addition to the X-linked locus. These 76 mice analyzed for M31 were also scored for restriction fragment length variants (RFLVs) for a number of established loci on the distal half of the X Chr--DXSmhl20, Pgk-1 (Cavanna et al. 1988), DXPasl (Amar et al. 1985)--and, in addition, scored for the recently mapped amelogenin (Amg) locus (Chapman et al. 1991). Amg is one of the most distal loci on the mouse X Chr (Chapman et al. 1991), lying 7 cM distal to DXWas31 (Disteche et al. 1989). Figure 2 gives the haplotypes of the 76 mice and two-point map distances between adjacent probes are shown. The order of loci was determined by minimizing the number of observed recombinants. A single recombination event places Cbx-rsl proximal to Amg and another recombinant placed it distal to DXPasl (Fig. 2). This positions Cbx-rsI 1.3 - 1.3 cM distal to DXPasl and 1.3 -+ 1.3 cM proximal to Amg. The mapping position of Cbx-rsl was confirmed by analysis of a number of Chinese hamster/mouse cell hybrids carrying translocation breakpoints of the mouse X Chr (Avner et al. 1987; Fig. 3). Each cell hybrid carries a mouse X/autosome translocation product containing the proximal half of the X Chr. Three hybrids--B20C12, E11 and N15--were utilized, carrying mouse X Chr material proximal to the T16H, T14R1 and T6R1 breakpoints, respectively. All three breakpoints lie proximal to the DXPasl locus (Avner et al. 1987) and their approximate genetic positions are indicated in Fig. 2. Hybridization of the M31 cDNA probe to Chinese hamster DNA detected a number of bands, but no signal is observed at 4.5 kb. Hybridization of the M31 probe to Taq I-digested DNAs from each of the hybrids failed to detect the 4.5 kb (C2) X-linked C57BL/10 allele indicating that Cbx-rsl maps distal to the T16H, T14R1 and T6R1 breakpoints, in agreement with the genetic mapping data. Previous studies have shown that the X-inactivation center in mouse maps distal to the T16H break-

74

R.M.J. Hamvas et al.: Mapping mouse homolog to Chr X

DXSmh 120 T16H T14R1 - - ~ T6R1

Pgk- I DXPas 1 Cbx- rs 1 Amg

i-1 m

I-1 B mN Nm mN l--lm mCl DimlY]ill 22

33

5

2

i-1 II! Nm mN Ji-I 8

4

i-1 N N m] m

I-I N N

1

1

IN

m

15.8+4.2

1.3+1.3

Numbers of backcross progeny t o t a l n u m b e r = 76

m

[]

= M.

spretus

r--] = 0 5 7 B L / 1 0 Fig. 2. Haplotype analysis and mapping of the mouse Cbx-rsl locus. Mice derived from the mdx interspecific backcross (Cavanna et al. 1988; this paper) and carrying recombination breakpoints distributed along the mouse X Chr have been analyzed with a variety of probes from the mouse X Chr. Individual backcross progeny mice were scored with a number of probes from the distal half of the X Chr for M. spretus (shaded boxes) and C57BL/10 (clear boxes) RFLVs. Locus order was determined by minimizing the number of recombination breakpoints. Probe details for DXSmhl20 and Pgk-1 have been described previously for this cross (Cavanna et al. 1988). The microclone, DXSmhl20 (Cavanna et al. 1988), detects a 3.4 kb Taq I RFLV in C57BL/10 and a 4 kb Taq I RFLV in M. spretus. The Pgk-1 probe, Pgk (Cavanna et al. 1988), detects a number of bands

and was scored using a 1.7 kb C57BL/10 Taq I RFLV and a 2.0 kb M. spretus Taq I RFLV. DXPasl (Amar et al. 1985) was scored using the probe p45 (a kind gift from P. Avner) which detects a 5 kb Taq I band in C57BL/10 and 2.4 kb Taq I band in M. spretus. The amelogenin locus (Amg) was scored using the probe pMA5.5, which detects a 9.0 kb Pst I band in C57BL/10 and a 14.0 kb Pst I band in M. spretus. In total, 76 mice were scored from the mdx backcross with all the indicated probes and include a number of mice that were not analyzed in the original work (Cavanna et al. 1988). The approximate positions of the T16H, T14R1 and T6R1 breakpoints are indicated with respect to the genetic map (Avner et al. 1987). In addition, interlocus distances (in cM) with standard errors are indicated.

point, proximal to the T14R1 and T6R1 breakpoints, a n d c l o s e t o t h e Pgk-1 l o c u s ( K e e r et al. 1990; R a s t a n a n d B r o w n 1990). F r o m o u r a n a l y s i s , h o w e v e r , t h e C b x - r s l l o c u s c l e a r l y m a p s d i s t a l to t h e X - i n a c t i v a t i o n region. We do not yet know whether Cbx-rsI represents a functional locus or not. However, given the g e n e t i c p o s i t i o n o f C b x - r s l , it is u n l i k e l y to p l a y a p r i m a r y r o l e in t h e i n i t i a t i o n o f X - i n a c t i v a t i o n .

Acknowledgments. This work was partly supported by grant No. G8803031CB from the Medical Research Council, UK to S.D.M. Brown. W. Reik is a Lister Institute Fellow. P.B. Singh is a Baraham Research Fellow. Part of this work was also supported by a grant from Combat Huntington's Chorea to W. Reik.

Fig. 3. Mapping of Cbx-rsl using somatic cell hybrids. All lanes are Taq I-digested DNAs hybridized to the M31 cDNA probe as described--see Fig. 1. Lane 1: C57BL/10; Lane 2: M. spretus; Lane 3: Chinese hamster; Lanes 4-6: hybrids containing X-autosome translocation breakpoints, T16H (B20C12), T14R1 ( E l l ) and T6R1 (Nt5)--see text. Lane 4: B20C12; Lane 5: E l l ; Lane 6: NIS. The X-linked 4.5 kb C57BL/10 allele is not observed in any hybrid.

References Amar, L.C., Arnaud, D., Cambrou, J.L., Gu6net, J.-L. and Avner, P.R.: Mapping of the mouse X chromosome using random genomic probes and an interspecific cross. EMBO J 4: 3695-3970, 1985. Avner, P., Arnaud, D., Amar, L., Cambrou, J., Winking, H. and Russell, L.B.: Characterisation of a panel of somatic cell hybrids for regional mapping of the mouse X chromosome. Proc NatI Acad Sci USA 84: 5330-5334, 1987. Brockdorff, N., Kay, G., Smith, S., Keer, J.T., Hamvas, R.M.J., Brown, S.D.M. and Rastan, S.: High-density molecular map of the central span of the mouse X chromosome. Genomics 10: 1722, 1991. Brown, S.D.M.: The X-inactivation center and mapping of the mouse X chromosome. In K. Davies and S. Tilghman (eds.); Genome Analysis: Gene Expression and its Control, Vol. 2, Cold Spring Harbor Laboratory Press, New York, in press, 1991. Cavanna, J.S., Coulton, G., Morgan, J.E., Brockdorff, N., Forrest, S.M., Davies, K.E. and Brown, S.D.M.: Molecular and genetic mapping of the mouse mdx locus. Genomics 3: 337-341, 1988. Chapman, V.M., Keitz, B.T., Disteche, C.M., Lau, E.C. and Snead, L.M.: Linkage of amelogenin (Amel) to the distal portion of the mouse X chromosome. Genomics 10: 23-28, 1991. Disteche, C.M., McConnell, G.K., Grant, S.G., Stephenson, D.A., Chapman, V.M., Gandy, S. and Adler, D.A.: Comparison of the physical and recombination maps of the mouse X chromosome. Genomics 5: 177-184, 1989. Eissenberg, J.C.: Position effect variegation in Drosophila: Towards a genetics of chromatin assembly. Bioessays 11: 14-17, I989. Eissenberg, J.C., James, T.C., Foster-Hartnett, D.M., Hartnett, T.,

R.M.J. Hamvas et al.: Mapping mouse homolog to Chr X Ngan, V. and Elgin, S.C.R.: Mutation in a heterochromatinspecific chromosomal protein is associated with suppressor of position-effect variegation in Drosophila melanogaster. Proc Nail Acad Sci USA 87: 9923-9927, 1991. Irving, N.G., Hardy, J.A. and Brown, S.D.M.: The multipoint genetic mapping of mouse chromosome 16. Genomics 9: 386-389, 1991. James, T.C. and Elgin, S.C.R.: Identification of a non-histone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene. Mol Cell Biol 6: 3862--3872, 1986. Keer, J.T., Hamvas, R.M.J., Brockdorff, N., Page, D., Rastan, S. and Brown, S.D.M.: Genetic mapping in the region of the mouse X-inactivation center. Genomics 7: 566-572, 1990. Lyon, M.F.: The quest for the X-inactivation center. Trends Genet 7: 69-70, 1991.

75 Paro, R.: Imprinting a determined state into the chromatin of Drosophila. Trends Genet 6: 416-421, 1990. Paro, R. and Hogness, D.S.: The Polycomb protein shares a homologous domain with a heterochromatin-associated prolein of Drosophila. Proc Natt Aead Sci USA 88: 263-267, 1991. Singh, P.B., Miller, J.R., Pearce, J., Kothary, R., Burton, R.D., Paro, R., James, T.C. and Gaunt, S.J.: A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants. Nucl Acids Res 19: 789-794, 1991. Spofford, J.: Position effect variegation in Drosophila. In M. Ashburner and E. Novitski (eds.); The Genetics and Biology o f Drosophila, pp. 955-1018, Academic Press, New York, 1976. Rastan, S. and Brown, S.D.M.: The search for the mouse X-chromosome inactivation center. Genet Res 56: 99-106, 1990.

Mapping of a mouse homolog of a heterochromatin protein gene the X chromosome.

Modifiers of position-effect-variegation in Drosophila are thought to encode proteins that are either structural components of heterochromatin or enzy...
520KB Sizes 0 Downloads 0 Views