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Short Communications Mammalian Genome 3: 650-652, 1992
9 Springer-Vedag New York Inc. 1992
Mapping of two human homologs of a Drosophila heterochromatin protein gene to the X Chromosome Wolf Reik, 1 Margaret A. Leversha, 2 Nick R. Waterfield, 1 and Prim B. Singh 1 1Department of Molecular Embryology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, UK; ZDepartment of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK Received May 13, 1992; accepted June 12, 1992
Position-effect variegation (PEV) is, in essence, the regional suppression of euchromatic genes brought into close proximity to heterochromatin by chromosomal rearrangement (Spofford 1976). A renewed interest has recently been shown in genetic modifiers of such variegating position effects (reviewed in Eissenberg 1989). The products of these genes are believed to change the local chromatin structure, leading to an increased (suppressor of variegation) or a decreased (enhancer) transcriptional activity. Cloning and characterization of one modifier gene, HPI, has shown it to be allelic to Suvar(2)5 (Eissenberg et al. 1990) and to be a structural component of heterochromatin (James and Elgin 1986). Regulation of gene expression by changes in chromatin structure has also been invoked as a mechanism for the maintenance of homeogene activity (Gaunt and Singh 1990; Paro 1990). In this system, heritable repression of homeogenes is brought about by a family of trans-regulators known as the Polycomb-group (Pcgroup). Interestingly, cloning and sequencing the group's namesake, Pc, has revealed a region of homology with HP1 (Paro and Hogness 1991). We have used this region, termed chromo box, to clone mammalian cDNAs that show significant homology to Drosophila HPI (Singh et al. 1991). One of these cDNAs, HSM1, is a human clone and shows 51% overall homology, at the amino acid level, with HP1 and 70% homology over the chromo domain. We have suggested that the mammalian HPl-like genes might function as gene repressors, perhaps by serving as components of heterochromatin. The classical example of this type of gene repression, in mammals, is X-Chromosome (Chr) inactivation (Lyon 1991). Our attention has, therefore, been focused on
Offprint requests to: W. Reik
the potential role of these genes in this process. This communication describes the chromosomal localization of two human X-linked homologs of the Drosophila HP1 gene. The chromosomal assignments of HSMXS and HSMXL were determined by dosage Southern blot
Fig. 1. Dosage hybridization of HSMl-related sequences. Peripheral blood DNA from normal males and females (10 wg each) was restricted with EcoRI, electrophoresed through a 0.8% agarose gel, and the blot was hybridized with the radiolabeled HSM1 cDNA probe under stringent conditions. Sizes of fragments are in kb.
W. Reik et al.: Human homologs of Drosophila gene analysis. Equal amounts of male and female D N A were digested with the restriction e n z y m e E c o R I , blotted, and probed with labeled HSM1 (Fig. I). At least six bands are detectable, four of which map to different autosomal locations (manuscript in preparation). Two bands, however, of 7.5 kb and 9 kb respectively, showed twice the intensity in female D N A compared with male D N A , suggesting that these two bands were located on the X Chr (Fig. 1). Two sets of overlapping cosmid clones were isolated from a human X Chr cosmid library (provided by H. Lehrach, I C R F London), using the HSM1 c D N A as a probe. Two cosmids (Bll61 and G01157) that contain the 7.5 kb and 9 kb E c o R I fragments were selected for further study, and the E c o R I fragments containing the sequences homologous to HSM1 were further subcloned into plasmids XS and XL. The regional localization of these clones was determined by in situ hybridization to prometaphase and metaphase c h r o m o s o m e s , using biotinylated probes (Fig. 2). Cosmid B1161 and plasmid subclone XS were m a p p e d on R - b a n d e d m e t a p h a s e c h r o m o s o m e s to Xp22.1, and this l o c a t i o n was f u r t h e r refined to Xp22.13 by hybridization to prometaphase chromosomes (Fig. 2A). This locus is designated H S M X S . Cosmid G01157 and plasmid X L were m a p p e d to Xql 1.2-12, just below the centromere (Fig. 2B). This locus is designated H S M X L . U n d e r slightly reduced stringency a proportion of metaphases also showed a signal at 17q 21.3, consistent with one of the autosomal genes mapping to this region (manuscript in preparation). Hence, H S M X S is likely to be homologous to the mouse gene C b x - r s l (Hamvas et al. 1992) as the human and m o u s e regions are syntenic. H S M X L might have been acquired at a later evolutionary stage, be-
Fig. 2. Localization of HSMXS and HSMXL by in situ hybridization. (A) R-banded normal male prometaphase chromosomes showing localization of cosmid Bll61 to Xp22.13. The same result was obtained with plasmid XS. (B) R-banded metaphase showing localization of cosmid G01157 to Xqll.2-12. The same result was obtained with plasmid XL. In situ hybridization with biotinylated cosmid and plasmid DNA was performed essentially as described by Kievits and colleagues (1990), using the hybridization buffer described by Viegas-Pequignot and co-workers (1989). Chromosome banding was visualized with a mixture of DAPI and propidium iodide according to Fan and colleagues (1990). Metaphases were examined with a Nikon Optiphot epifluorescence microscope, and images of banded metaphases with superimposed hybridization signals were collected with an MRC Lasersharp 600 confocal microscope (Bio-Rad).
651 cause there is no mouse homolog of this gene. Both genes map well outside the X inactivation region and Xist (Brown et al. 1991) and are therefore unlikely to play any primary role in the initiation of X Chr inactivation. It is interesting to note that two recent models for the parental effect in Huntington's Chorea propose that dosage-dependent genes on the X Chr can modify expression of the disease locus (Laird 1990; Sapienza 1990). Preliminary sequence analysis of plasmids XS and X L indicates that both genes might be processed retroposons (Weiner et al. 1986; unpublished) and it will be interesting to see whether they are expressed and whether they encode any functional products.
Acknowledgments. P.B. Singh is a Babraham Research Fellow; W.
Reik is a Fellow of the Lister Institute of Preventive Medicine. Part of this work was supported by a grant from Combat Huntington's Chorea. We thank G. Zehetner and H. Lehrach for help with screening the X Chr cosmid library.
References Brown, C.F., Lafreuiere, R.G., Powers, V.E., Sebastio, G., Ballabio, A., Pettigrew, A.L., Ledbetter, D.H., Levy, E., Craig, I.W., and Willard, H.F.: Localization of the X inactivation centre on the human X chromosome in Xql3. Nature 349: 82-84, 1991. Eissenberg, J.C.: Position effect variegation in Drosophila: towards a genetics of chromatin assembly. Bioessays 11: 14-17, 1989. Eissenberg, J.C., James, T.C., Foster-Hartnett, D.M., Hartnett, T., 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 Natl Acad Sci USA 87: 9923-9927, 1990. Fan, Y.-S., Davis, L.M., and Shows, T.B.: Mapping small DNA sequences by fluorescencein situ hybridization onto banded chromosomes. Proc Natl Acad Sci USA 87: 6223-6227, 1990. Gaunt, S.J. and Singh, P.B.: Homeogene expression patterns and chromosomal imprinting. Trends Genet 6:208-212, 1990. Hamvas, R.M.J., Reik, W., Gaunt, S.J., Brown, S.D.M., and Singh, P.B.: Mapping of a mouse homolog of a Heterochromatin protein gene to the X Chromosome. Mammalian Genome 2: 7275, 1992. 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. Kievits, T., Dauwerse, J.G., Wiegant, J., Devilee, P., Breuning, M.H., Cornelisse, C.J., van Ommen G.-J.B., and Pearson, P.L.: Rapid subchromosomal localization of cosmids by non-radioactive in situ hybridization. Cytogenet Cell Genet 53: 134-136, 1990. Laird, C.D.: Proposed genetic basis of Huntington's disease. Trends Genet 6: 242-247, 1990. Lyon, M.F.: The quest for the X-inactivation center. Trends Genet 7: 69-70, 1991. Paro, R.: Imprintinga determined state into the chromatin of Drosophila melanogaster. Trends Genet 6: 416--421, 1990. Paro, R. and Hogness, D.S.: The polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. Proc Natl Acad Sci USA 88: 263-267, 1991. Sapienza, C.: Sex-linked dosage-sensitive modifiers as imprinting genes. Development (Suppl): 107-114, 1990. Singh, P.B., Miller, J.R., Pearce, J., Kothary, R., Burton, R.D.,
652 Paro, R., James, T.C., and Gaunt, S.J.: A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants. Nucleic Acids Res 19: 789-794, 1991. Spofford, J.: Position effect variegation in Drosophila. In M. Ashburner and E. Novitski (eds.); The Genetics and Biology of Drosophila, pp. 955-1018, Academic Press, New York, 1976. Viegas-Pequignot, E., Dutrillaux, B., Magdelenat, H., and CoppeyMoisan, M.: Mapping of single-copy DNA sequences on human
W. Reik et al.: Human homologs of Drosophila gene chromosomes by in situ hybridization with biotinylated probes: enhancement of detection sensitivity by intensified-fluorescence digital-imagingmicroscopy. Proc Natl Acad Sci USA 86: 582-586, 1989. Weiner, A.M., Deininger, P.L., and Efstratiadis, A.: Nonviral retroposons: genes, pseudogenes and transposable elements generated by the reverse flow of genetic information. Annu Rev Biochem 55: 631--661, 1986.
Short Communications Mammalian Genome 3: 650-652, 1992
9 Springer-Vedag New York Inc. 1992
Mapping of two human homologs of a Drosophila heterochromatin protein gene to the X Chromosome Wolf Reik, 1 Margaret A. Leversha, 2 Nick R. Waterfield, 1 and Prim B. Singh 1 1Department of Molecular Embryology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, UK; ZDepartment of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK Received May 13, 1992; accepted June 12, 1992
Position-effect variegation (PEV) is, in essence, the regional suppression of euchromatic genes brought into close proximity to heterochromatin by chromosomal rearrangement (Spofford 1976). A renewed interest has recently been shown in genetic modifiers of such variegating position effects (reviewed in Eissenberg 1989). The products of these genes are believed to change the local chromatin structure, leading to an increased (suppressor of variegation) or a decreased (enhancer) transcriptional activity. Cloning and characterization of one modifier gene, HPI, has shown it to be allelic to Suvar(2)5 (Eissenberg et al. 1990) and to be a structural component of heterochromatin (James and Elgin 1986). Regulation of gene expression by changes in chromatin structure has also been invoked as a mechanism for the maintenance of homeogene activity (Gaunt and Singh 1990; Paro 1990). In this system, heritable repression of homeogenes is brought about by a family of trans-regulators known as the Polycomb-group (Pcgroup). Interestingly, cloning and sequencing the group's namesake, Pc, has revealed a region of homology with HP1 (Paro and Hogness 1991). We have used this region, termed chromo box, to clone mammalian cDNAs that show significant homology to Drosophila HPI (Singh et al. 1991). One of these cDNAs, HSM1, is a human clone and shows 51% overall homology, at the amino acid level, with HP1 and 70% homology over the chromo domain. We have suggested that the mammalian HPl-like genes might function as gene repressors, perhaps by serving as components of heterochromatin. The classical example of this type of gene repression, in mammals, is X-Chromosome (Chr) inactivation (Lyon 1991). Our attention has, therefore, been focused on
Offprint requests to: W. Reik
the potential role of these genes in this process. This communication describes the chromosomal localization of two human X-linked homologs of the Drosophila HP1 gene. The chromosomal assignments of HSMXS and HSMXL were determined by dosage Southern blot
Fig. 1. Dosage hybridization of HSMl-related sequences. Peripheral blood DNA from normal males and females (10 wg each) was restricted with EcoRI, electrophoresed through a 0.8% agarose gel, and the blot was hybridized with the radiolabeled HSM1 cDNA probe under stringent conditions. Sizes of fragments are in kb.
W. Reik et al.: Human homologs of Drosophila gene analysis. Equal amounts of male and female D N A were digested with the restriction e n z y m e E c o R I , blotted, and probed with labeled HSM1 (Fig. I). At least six bands are detectable, four of which map to different autosomal locations (manuscript in preparation). Two bands, however, of 7.5 kb and 9 kb respectively, showed twice the intensity in female D N A compared with male D N A , suggesting that these two bands were located on the X Chr (Fig. 1). Two sets of overlapping cosmid clones were isolated from a human X Chr cosmid library (provided by H. Lehrach, I C R F London), using the HSM1 c D N A as a probe. Two cosmids (Bll61 and G01157) that contain the 7.5 kb and 9 kb E c o R I fragments were selected for further study, and the E c o R I fragments containing the sequences homologous to HSM1 were further subcloned into plasmids XS and XL. The regional localization of these clones was determined by in situ hybridization to prometaphase and metaphase c h r o m o s o m e s , using biotinylated probes (Fig. 2). Cosmid B1161 and plasmid subclone XS were m a p p e d on R - b a n d e d m e t a p h a s e c h r o m o s o m e s to Xp22.1, and this l o c a t i o n was f u r t h e r refined to Xp22.13 by hybridization to prometaphase chromosomes (Fig. 2A). This locus is designated H S M X S . Cosmid G01157 and plasmid X L were m a p p e d to Xql 1.2-12, just below the centromere (Fig. 2B). This locus is designated H S M X L . U n d e r slightly reduced stringency a proportion of metaphases also showed a signal at 17q 21.3, consistent with one of the autosomal genes mapping to this region (manuscript in preparation). Hence, H S M X S is likely to be homologous to the mouse gene C b x - r s l (Hamvas et al. 1992) as the human and m o u s e regions are syntenic. H S M X L might have been acquired at a later evolutionary stage, be-
Fig. 2. Localization of HSMXS and HSMXL by in situ hybridization. (A) R-banded normal male prometaphase chromosomes showing localization of cosmid Bll61 to Xp22.13. The same result was obtained with plasmid XS. (B) R-banded metaphase showing localization of cosmid G01157 to Xqll.2-12. The same result was obtained with plasmid XL. In situ hybridization with biotinylated cosmid and plasmid DNA was performed essentially as described by Kievits and colleagues (1990), using the hybridization buffer described by Viegas-Pequignot and co-workers (1989). Chromosome banding was visualized with a mixture of DAPI and propidium iodide according to Fan and colleagues (1990). Metaphases were examined with a Nikon Optiphot epifluorescence microscope, and images of banded metaphases with superimposed hybridization signals were collected with an MRC Lasersharp 600 confocal microscope (Bio-Rad).
651 cause there is no mouse homolog of this gene. Both genes map well outside the X inactivation region and Xist (Brown et al. 1991) and are therefore unlikely to play any primary role in the initiation of X Chr inactivation. It is interesting to note that two recent models for the parental effect in Huntington's Chorea propose that dosage-dependent genes on the X Chr can modify expression of the disease locus (Laird 1990; Sapienza 1990). Preliminary sequence analysis of plasmids XS and X L indicates that both genes might be processed retroposons (Weiner et al. 1986; unpublished) and it will be interesting to see whether they are expressed and whether they encode any functional products.
Acknowledgments. P.B. Singh is a Babraham Research Fellow; W.
Reik is a Fellow of the Lister Institute of Preventive Medicine. Part of this work was supported by a grant from Combat Huntington's Chorea. We thank G. Zehetner and H. Lehrach for help with screening the X Chr cosmid library.
References Brown, C.F., Lafreuiere, R.G., Powers, V.E., Sebastio, G., Ballabio, A., Pettigrew, A.L., Ledbetter, D.H., Levy, E., Craig, I.W., and Willard, H.F.: Localization of the X inactivation centre on the human X chromosome in Xql3. Nature 349: 82-84, 1991. Eissenberg, J.C.: Position effect variegation in Drosophila: towards a genetics of chromatin assembly. Bioessays 11: 14-17, 1989. Eissenberg, J.C., James, T.C., Foster-Hartnett, D.M., Hartnett, T., 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 Natl Acad Sci USA 87: 9923-9927, 1990. Fan, Y.-S., Davis, L.M., and Shows, T.B.: Mapping small DNA sequences by fluorescencein situ hybridization onto banded chromosomes. Proc Natl Acad Sci USA 87: 6223-6227, 1990. Gaunt, S.J. and Singh, P.B.: Homeogene expression patterns and chromosomal imprinting. Trends Genet 6:208-212, 1990. Hamvas, R.M.J., Reik, W., Gaunt, S.J., Brown, S.D.M., and Singh, P.B.: Mapping of a mouse homolog of a Heterochromatin protein gene to the X Chromosome. Mammalian Genome 2: 7275, 1992. 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. Kievits, T., Dauwerse, J.G., Wiegant, J., Devilee, P., Breuning, M.H., Cornelisse, C.J., van Ommen G.-J.B., and Pearson, P.L.: Rapid subchromosomal localization of cosmids by non-radioactive in situ hybridization. Cytogenet Cell Genet 53: 134-136, 1990. Laird, C.D.: Proposed genetic basis of Huntington's disease. Trends Genet 6: 242-247, 1990. Lyon, M.F.: The quest for the X-inactivation center. Trends Genet 7: 69-70, 1991. Paro, R.: Imprintinga determined state into the chromatin of Drosophila melanogaster. Trends Genet 6: 416--421, 1990. Paro, R. and Hogness, D.S.: The polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. Proc Natl Acad Sci USA 88: 263-267, 1991. Sapienza, C.: Sex-linked dosage-sensitive modifiers as imprinting genes. Development (Suppl): 107-114, 1990. Singh, P.B., Miller, J.R., Pearce, J., Kothary, R., Burton, R.D.,
652 Paro, R., James, T.C., and Gaunt, S.J.: A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants. Nucleic Acids Res 19: 789-794, 1991. Spofford, J.: Position effect variegation in Drosophila. In M. Ashburner and E. Novitski (eds.); The Genetics and Biology of Drosophila, pp. 955-1018, Academic Press, New York, 1976. Viegas-Pequignot, E., Dutrillaux, B., Magdelenat, H., and CoppeyMoisan, M.: Mapping of single-copy DNA sequences on human
W. Reik et al.: Human homologs of Drosophila gene chromosomes by in situ hybridization with biotinylated probes: enhancement of detection sensitivity by intensified-fluorescence digital-imagingmicroscopy. Proc Natl Acad Sci USA 86: 582-586, 1989. Weiner, A.M., Deininger, P.L., and Efstratiadis, A.: Nonviral retroposons: genes, pseudogenes and transposable elements generated by the reverse flow of genetic information. Annu Rev Biochem 55: 631--661, 1986.
