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Chromosomal Assignment of HumanID1andID2Genes
SHORT COMMUNICATION Chromosomal Assignment of Human ID1 and ID2 Genes SUSAN MATHEW,* WEIYI CHEN,* VUNDAVALLI V. V. S. MURTY,* ROBERT BENEZRA,* AND R. S. K. CHAGANTI*,†,1 †Department of Human Genetics and the *Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021 Received August 14, 1995; accepted September 7, 1995
The Id (inhibitor of DNA binding) proteins regulate transcription during development by interacting with transcription factors. Three human genes, ID1, ID2, and ID3, have been identified that belong to this family of transcription regulators. We show, by somatic cell hybridization and fluorescence in situ hybridization experiments, that ID1 and ID2 are localized at 20q11 and 2p25, respectively. q 1995 Academic Press, Inc.
The Id (inhibitor of DNA binding) proteins are members of the basic helix – loop – helix (bHLH) protein family that act as negative regulators of other members of the family (2). They retain the ability to dimerize, but lack the basic DNA binding domain, and they interact with some bHLH proteins such as MyoD, E12, and E47 and inhibit their DNA binding in vitro as well as in vivo (2, 8), but do not interact with other bHLH proteins such as MYC, TFE3, USF, and AP4 (14). Because they interact with transcription factors, the Id proteins are suggested to regulate coordinated timing of transcriptional regulation during development (14). Id mRNA is downregulated in a variety of cell types when induced to undergo differentiation. Thus, downregulation of Id protein enables MyoD and E2A proteins to bind DNA and to activate the cascade of gene transcription leading to differentiation. The Id proteins are expressed in most tissues of the early embryo, and this expression disappears as embryonic development proceeds (15). Id proteins appear to inhibit differentiation either through inhibition of E proteins or through inhibition of heterodimeric complexes from binding DNA and transactivating target genes (8, 11). In the mouse, three members of the Id family have been identified, namely Id1, Id2, (2, 14), and HLH462.3, which is also referred to as Id3 (4). The HLH domain in all three genes is highly conserved. 1
To whom correspondence should be addressed.
Previous mapping results indicated that the murine Id1, Id2, and Id3 genes are located on chromosomes 2, 12, and 4, respectively (14). The chromosomal assignment of the human homologs has not been determined. In this report, based on somatic cell hybridization and fluorescence in situ hybridization (FISH) analyses, we show that the human ID1 and ID2 genes are localized to 20q11 and 2p25, respectively. Somatic cell hybrid analysis of ID1 was performed using a panel of DNAs from 27 cell lines containing various human chromosomes against a hamster background (Bios Laboratories, New Haven, CT). NIGMS human/rodent somatic cell hybrid mapping panel 2 (Coriell Institute for Medical Research, NJ) was used for assigning the ID2 gene. The plasmid pId1 containing a partial human ID1 cDNA and a complete human ID2 cDNA cloned into pBluescript was used as the hybridization probe (3). To localize the ID genes subregionally to chromosomes, FISH was performed on metaphase chromosome spreads from PHA-stimulated peripheral blood lymphocytes. Phage clones containing ID1 and ID2 genomic sequences were used as FISH probes. These probes were obtained by screening a human genomic library constructed from partial Sau3A-digested placental DNA cloned into l GEM-11 with the cDNA probes. Two clones with insert sizes of 15 and 17 kb containing ID1 and ID2 sequences, respectively, were isolated. For FISH, the phages were nick-translated with biotin-11 – dUTP (BRL, Gaithersburg, MD). Hybridization and detection of labeled probes were performed as previously described (12). Separate images of DAPI-stained chromosomes and hybridization signals were captured by a cooled chargecoupled device camera (Photometrics, Tucson, AZ) and analyzed using the Smartcapture Image Analysis System (Digital Scientific, Cambridge). We assigned the ID1 gene to chromosome 20 by hybridizing the plasmid containing the partial cDNA to Southern blots of a PstI-digested somatic cell hybrid DNA GENOMICS
385
/ m4534$3764
10-26-95 02:24:11
gnmas
30, 385–387 (1995)
0888-7543/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
AP-Genomics
386
SHORT COMMUNICATION
panel. The probe showed signals syntenic with chromosome 20, with 100% concordance (data not shown). FISH was then used to refine the chromosomal localization of the ID1 gene; a total of 39 metaphases with hybridization signals were analyzed. The signals clustered on band 20q11. No specific double signals were observed on any other chromosomal regions, thus allowing the precise localization of the ID1 gene to 20q11. (Fig. 1). Southern blot analysis of the ID2 gene on the PstIdigested monochromosomal somatic cell hybrid panel gave signals with the NA10826B hybrid containing human chromosome 2 (data not shown). A total of 50 metaphases with FISH signals were analyzed. Specific hybridization signals were clustered at 2p25, thus allowing subregional assignment of ID2 to 2p25 (Fig. 2). Id proteins belong to an expanding family of eukaryotic transcriptional regulators characterized by the presence of a highly conserved HLH motif, which mediates homo- and heterodimerization (9, 10). Id genes are strongly expressed in undifferentiated proliferating and tumor cells. They can be induced upon stimulation with serum, growth factors, or phorbol esters but are downregulated in quiescent cells or during differentiation (1 – 5, 8, 11). Id family proteins are suggested to function as general inhibitors of differentiation (7) and controllers of growth induction. Also, the Id proteins are required for progression through the G1 phase (6) and are involved in regulatory events prior to or near the restriction points in the G1 phase of the cell cycle, possibly by antagonizing the growth arrest mediated by the E proteins (13). The determination of the chromosomal position of these genes in the human genome is important because of their role in cell proliferation and differentiation.
FIG. 1. Localization of ID1 gene by fluorescence in situ hybridization. (A) Partial metaphase showing specific site of hybridization of the ID1 gene. Arrow indicates FISH signal. (B) Partial metaphase showing the DAPI-stained chromosome bands. Arrowhead indicates band 20q11. (C) Idiogram of the human G-banded chromosome 20, illustrating the site of the ID1 gene hybridization. Each large star represents 10 double signals (on both chromatids). Each small star represents one double signal, and the small circle represents one single signal.
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FIG. 2. Localization of ID2 gene by fluorescence in situ hybridization. (A) Partial metaphase showing specific site of hybridization of the ID2 gene. Arrow indicates FISH signal. (B) Partial metaphase showing the DAPI-stained chromosome bands. Arrowhead indicates band 2p25. (C) Idiogram of the human G-banded chromosome 2, illustrating the site of the ID2 gene hybridization. Each large star represents 10 double signals (on both chromatids), and each large circle represents 10 single (on one chromatid) signals. Small stars and circles represent one double and one single signal, respectively.
ACKNOWLEDGMENTS This investigation was supported by NIH Grant CA-34775 (R.S.K.C.) and NSF Grant IBN-9118977 (R.B.).
REFERENCES 1. Barone, M. V., Pepperkok, R., Peverali, F. A., and Philipson, L. (1994). Id proteins control growth induction in mammalian cells. Proc. Natl. Acad. Sci. USA 91: 4985–4988. 2. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990). The protein Id: A negative regulator of helix– loop–helix DNA binding proteins. Cell 61: 49–59. 3. Biggs, J., Murphy, E. V., and Israel, M. A. (1992). A human Idlike helix–loop–helix protein expressed during early development. Proc. Natl. Acad. Sci. USA 89: 1512–1516. 4. Christy, B. A., Sanders, L. K., Lau, L. F., Copeland, N. G., Jenkins, N. A., and Nathans, D. (1991). An Id-related helix–loop–helix protein encoded by a growth factor inducible gene. Proc. Natl. Acad. Sci. USA 88: 1815–1819. 5. Deed, R. W., Bianchi, S. M., Atherton, G. T., Johnston, D., Santibanez-Koref, M., Murphy, J. J., and Norton, J. D. (1993). An immediate early human gene encodes an Id-like helix–loop–helix protein and is regulated by protein kinase C activation in diverse cell types. Oncogene 8: 599–607. 6. Hara, E., Yamaguchi, T., Nojima, H., Ide, T., Campisi, J., Okayama, H., and Oda, K. (1994). Id-related genes encoding helix–loop– helix proteins are required for G1 progression and are repressed in senescent human fibroblasts. J. Biol. Chem. 269: 2139–2145. 7. Jan, Y. N., and Jan, L. Y. (1993). HLH proteins, fly neurogenesis, and vertebrate myogenesis. Cell 75: 827–830. 8. Jen, Y., Weintraub, H., and Benezra, R. (1992). Overexpression of Id protein inhibits the muscle differentiation program: In vivo association of Id with E2A proteins. Genes Dev. 6: 1466–1479. 9. Kadesch, T. (1992). Helix–loop–helix proteins in the regulation of immunoglobulin gene transcription. Immunol. Today 13: 31– 36.
AP-Genomics
SHORT COMMUNICATION 10. Kadesch, T. (1993). Consequences of heteromeric interactions among helix–loop–helix proteins. Cell Growth Differ. 4: 49–55. 11. Kreider, B. L., Benezra, R., Rovera, G., and Kadesch, T. (1992). Inhibitor of myeloid differentiation by the helix–loop–helix protein Id. Science (Washington DC) 255: 1700–1702. 12. Mathew, S., Murty, V. V. V. S., Hunziker, W., and Chaganti, R. S. K. (1992). Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in situ hybridization. Genomics 14: 775–778.
/ m4534$3764
10-26-95 02:24:11
gnmas
387
13. Peverali, F. A., Ramqvist, T., Saffrich, R., Pepperkok, R., Barone, M. V., Philipson, L. (1994). Regulation of G1 expression of E2A and Id helix–loop–helix proteins. EMBO J. 13: 4291–4301. 14. Sun, X-H., Copeland, N. G., Jenkins, N. A., and Baltimore, D. (1991). Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix–loop–helix proteins. Mol. Cell. Biol. 11: 5603– 5611. 15. Wang, Y., Benezra, R., and Sassoon, D. A. (1992). Id expression during mouse development: A role in morphogenesis. Dev. Dyn. 94: 222–230.
AP-Genomics
The Id (inhibitor of DNA binding) proteins regulate transcription during development by interacting with transcription factors. Three human genes, ID1, ID2, and ID3, have been identified that belong to this family of transcription regulators. We show, by somatic cell hybridization and fluorescence in situ hybridization experiments, that ID1 and ID2 are localized at 20q11 and 2p25, respectively. q 1995 Academic Press, Inc.
The Id (inhibitor of DNA binding) proteins are members of the basic helix – loop – helix (bHLH) protein family that act as negative regulators of other members of the family (2). They retain the ability to dimerize, but lack the basic DNA binding domain, and they interact with some bHLH proteins such as MyoD, E12, and E47 and inhibit their DNA binding in vitro as well as in vivo (2, 8), but do not interact with other bHLH proteins such as MYC, TFE3, USF, and AP4 (14). Because they interact with transcription factors, the Id proteins are suggested to regulate coordinated timing of transcriptional regulation during development (14). Id mRNA is downregulated in a variety of cell types when induced to undergo differentiation. Thus, downregulation of Id protein enables MyoD and E2A proteins to bind DNA and to activate the cascade of gene transcription leading to differentiation. The Id proteins are expressed in most tissues of the early embryo, and this expression disappears as embryonic development proceeds (15). Id proteins appear to inhibit differentiation either through inhibition of E proteins or through inhibition of heterodimeric complexes from binding DNA and transactivating target genes (8, 11). In the mouse, three members of the Id family have been identified, namely Id1, Id2, (2, 14), and HLH462.3, which is also referred to as Id3 (4). The HLH domain in all three genes is highly conserved. 1
To whom correspondence should be addressed.
Previous mapping results indicated that the murine Id1, Id2, and Id3 genes are located on chromosomes 2, 12, and 4, respectively (14). The chromosomal assignment of the human homologs has not been determined. In this report, based on somatic cell hybridization and fluorescence in situ hybridization (FISH) analyses, we show that the human ID1 and ID2 genes are localized to 20q11 and 2p25, respectively. Somatic cell hybrid analysis of ID1 was performed using a panel of DNAs from 27 cell lines containing various human chromosomes against a hamster background (Bios Laboratories, New Haven, CT). NIGMS human/rodent somatic cell hybrid mapping panel 2 (Coriell Institute for Medical Research, NJ) was used for assigning the ID2 gene. The plasmid pId1 containing a partial human ID1 cDNA and a complete human ID2 cDNA cloned into pBluescript was used as the hybridization probe (3). To localize the ID genes subregionally to chromosomes, FISH was performed on metaphase chromosome spreads from PHA-stimulated peripheral blood lymphocytes. Phage clones containing ID1 and ID2 genomic sequences were used as FISH probes. These probes were obtained by screening a human genomic library constructed from partial Sau3A-digested placental DNA cloned into l GEM-11 with the cDNA probes. Two clones with insert sizes of 15 and 17 kb containing ID1 and ID2 sequences, respectively, were isolated. For FISH, the phages were nick-translated with biotin-11 – dUTP (BRL, Gaithersburg, MD). Hybridization and detection of labeled probes were performed as previously described (12). Separate images of DAPI-stained chromosomes and hybridization signals were captured by a cooled chargecoupled device camera (Photometrics, Tucson, AZ) and analyzed using the Smartcapture Image Analysis System (Digital Scientific, Cambridge). We assigned the ID1 gene to chromosome 20 by hybridizing the plasmid containing the partial cDNA to Southern blots of a PstI-digested somatic cell hybrid DNA GENOMICS
385
/ m4534$3764
10-26-95 02:24:11
gnmas
30, 385–387 (1995)
0888-7543/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
AP-Genomics
386
SHORT COMMUNICATION
panel. The probe showed signals syntenic with chromosome 20, with 100% concordance (data not shown). FISH was then used to refine the chromosomal localization of the ID1 gene; a total of 39 metaphases with hybridization signals were analyzed. The signals clustered on band 20q11. No specific double signals were observed on any other chromosomal regions, thus allowing the precise localization of the ID1 gene to 20q11. (Fig. 1). Southern blot analysis of the ID2 gene on the PstIdigested monochromosomal somatic cell hybrid panel gave signals with the NA10826B hybrid containing human chromosome 2 (data not shown). A total of 50 metaphases with FISH signals were analyzed. Specific hybridization signals were clustered at 2p25, thus allowing subregional assignment of ID2 to 2p25 (Fig. 2). Id proteins belong to an expanding family of eukaryotic transcriptional regulators characterized by the presence of a highly conserved HLH motif, which mediates homo- and heterodimerization (9, 10). Id genes are strongly expressed in undifferentiated proliferating and tumor cells. They can be induced upon stimulation with serum, growth factors, or phorbol esters but are downregulated in quiescent cells or during differentiation (1 – 5, 8, 11). Id family proteins are suggested to function as general inhibitors of differentiation (7) and controllers of growth induction. Also, the Id proteins are required for progression through the G1 phase (6) and are involved in regulatory events prior to or near the restriction points in the G1 phase of the cell cycle, possibly by antagonizing the growth arrest mediated by the E proteins (13). The determination of the chromosomal position of these genes in the human genome is important because of their role in cell proliferation and differentiation.
FIG. 1. Localization of ID1 gene by fluorescence in situ hybridization. (A) Partial metaphase showing specific site of hybridization of the ID1 gene. Arrow indicates FISH signal. (B) Partial metaphase showing the DAPI-stained chromosome bands. Arrowhead indicates band 20q11. (C) Idiogram of the human G-banded chromosome 20, illustrating the site of the ID1 gene hybridization. Each large star represents 10 double signals (on both chromatids). Each small star represents one double signal, and the small circle represents one single signal.
/ m4534$3764
10-26-95 02:24:11
gnmas
FIG. 2. Localization of ID2 gene by fluorescence in situ hybridization. (A) Partial metaphase showing specific site of hybridization of the ID2 gene. Arrow indicates FISH signal. (B) Partial metaphase showing the DAPI-stained chromosome bands. Arrowhead indicates band 2p25. (C) Idiogram of the human G-banded chromosome 2, illustrating the site of the ID2 gene hybridization. Each large star represents 10 double signals (on both chromatids), and each large circle represents 10 single (on one chromatid) signals. Small stars and circles represent one double and one single signal, respectively.
ACKNOWLEDGMENTS This investigation was supported by NIH Grant CA-34775 (R.S.K.C.) and NSF Grant IBN-9118977 (R.B.).
REFERENCES 1. Barone, M. V., Pepperkok, R., Peverali, F. A., and Philipson, L. (1994). Id proteins control growth induction in mammalian cells. Proc. Natl. Acad. Sci. USA 91: 4985–4988. 2. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990). The protein Id: A negative regulator of helix– loop–helix DNA binding proteins. Cell 61: 49–59. 3. Biggs, J., Murphy, E. V., and Israel, M. A. (1992). A human Idlike helix–loop–helix protein expressed during early development. Proc. Natl. Acad. Sci. USA 89: 1512–1516. 4. Christy, B. A., Sanders, L. K., Lau, L. F., Copeland, N. G., Jenkins, N. A., and Nathans, D. (1991). An Id-related helix–loop–helix protein encoded by a growth factor inducible gene. Proc. Natl. Acad. Sci. USA 88: 1815–1819. 5. Deed, R. W., Bianchi, S. M., Atherton, G. T., Johnston, D., Santibanez-Koref, M., Murphy, J. J., and Norton, J. D. (1993). An immediate early human gene encodes an Id-like helix–loop–helix protein and is regulated by protein kinase C activation in diverse cell types. Oncogene 8: 599–607. 6. Hara, E., Yamaguchi, T., Nojima, H., Ide, T., Campisi, J., Okayama, H., and Oda, K. (1994). Id-related genes encoding helix–loop– helix proteins are required for G1 progression and are repressed in senescent human fibroblasts. J. Biol. Chem. 269: 2139–2145. 7. Jan, Y. N., and Jan, L. Y. (1993). HLH proteins, fly neurogenesis, and vertebrate myogenesis. Cell 75: 827–830. 8. Jen, Y., Weintraub, H., and Benezra, R. (1992). Overexpression of Id protein inhibits the muscle differentiation program: In vivo association of Id with E2A proteins. Genes Dev. 6: 1466–1479. 9. Kadesch, T. (1992). Helix–loop–helix proteins in the regulation of immunoglobulin gene transcription. Immunol. Today 13: 31– 36.
AP-Genomics
SHORT COMMUNICATION 10. Kadesch, T. (1993). Consequences of heteromeric interactions among helix–loop–helix proteins. Cell Growth Differ. 4: 49–55. 11. Kreider, B. L., Benezra, R., Rovera, G., and Kadesch, T. (1992). Inhibitor of myeloid differentiation by the helix–loop–helix protein Id. Science (Washington DC) 255: 1700–1702. 12. Mathew, S., Murty, V. V. V. S., Hunziker, W., and Chaganti, R. S. K. (1992). Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in situ hybridization. Genomics 14: 775–778.
/ m4534$3764
10-26-95 02:24:11
gnmas
387
13. Peverali, F. A., Ramqvist, T., Saffrich, R., Pepperkok, R., Barone, M. V., Philipson, L. (1994). Regulation of G1 expression of E2A and Id helix–loop–helix proteins. EMBO J. 13: 4291–4301. 14. Sun, X-H., Copeland, N. G., Jenkins, N. A., and Baltimore, D. (1991). Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix–loop–helix proteins. Mol. Cell. Biol. 11: 5603– 5611. 15. Wang, Y., Benezra, R., and Sassoon, D. A. (1992). Id expression during mouse development: A role in morphogenesis. Dev. Dyn. 94: 222–230.
AP-Genomics