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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

Previous mapping results indicated that the mu-The Id (inhibitor of DNA binding) proteins regu- rine Id1, Id2, and Id3 genes are located on chromo-

late transcription during development by inter- somes 2, 12, and 4, respectively (14). The chromo-acting with transcription factors. Three human somal assignment of the human homologs has notgenes, ID1, ID2, and ID3, have been identified that been determined. In this report, based on somaticbelong to this family of transcription regulators. We cell hybridization and fluorescence in situ hybridiza-show, by somatic cell hybridization and fluores- tion (FISH) analyses, we show that the human ID1cence in situ hybridization experiments, that ID1 and ID2 genes are localized to 20q11 and 2p25, re-and ID2 are localized at 20q11 and 2p25, respec- spectively.tively. q 1995 Academic Press, Inc.

Somatic cell hybrid analysis of ID1 was performedusing a panel of DNAs from 27 cell lines containing

The Id (inhibitor of DNA binding) proteins are various human chromosomes against a hamstermembers of the basic helix– loop–helix (bHLH) pro- background (Bios Laboratories, New Haven, CT).tein family that act as negative regulators of other NIGMS human/rodent somatic cell hybrid mappingmembers of the family (2). They retain the ability panel 2 (Coriell Institute for Medical Research, NJ)to dimerize, but lack the basic DNA binding domain, was used for assigning the ID2 gene. The plasmidand they interact with some bHLH proteins such as pId1 containing a partial human ID1 cDNA and aMyoD, E12, and E47 and inhibit their DNA binding complete human ID2 cDNA cloned into pBluescriptin vitro as well as in vivo (2, 8), but do not interact was used as the hybridization probe (3).with other bHLH proteins such as MYC, TFE3, USF, To localize the ID genes subregionally to chromosomes,and AP4 (14). Because they interact with transcrip- FISH was performed on metaphase chromosome spreadstion factors, the Id proteins are suggested to regu- from PHA-stimulated peripheral blood lymphocytes.late coordinated timing of transcriptional regulation Phage clones containing ID1 and ID2 genomic se-during development (14). Id mRNA is downregu- quences were used as FISH probes. These probes werelated in a variety of cell types when induced to un- obtained by screening a human genomic library con-dergo differentiation. Thus, downregulation of Id structed from partial Sau3A-digested placentalprotein enables MyoD and E2A proteins to bind DNA cloned into l GEM-11 with the cDNA probes.DNA and to activate the cascade of gene transcrip- Two clones with insert sizes of 15 and 17 kb con-tion leading to differentiation. The Id proteins are taining ID1 and ID2 sequences, respectively, wereexpressed in most tissues of the early embryo, and isolated. For FISH, the phages were nick-translatedthis expression disappears as embryonic develop- with biotin-11–dUTP (BRL, Gaithersburg, MD).ment proceeds (15). Id proteins appear to inhibit dif- Hybridization and detection of labeled probes wereferentiation either through inhibition of E proteins performed as previously described (12). Separateor through inhibition of heterodimeric complexes

images of DAPI-stained chromosomes and hybrid-from binding DNA and transactivating target genesization signals were captured by a cooled charge-(8, 11).coupled device camera (Photometrics, Tucson, AZ)In the mouse, three members of the Id family haveand analyzed using the Smartcapture Image Analy-been identified, namely Id1, Id2, (2, 14), and HLH462.3,sis System (Digital Scientific, Cambridge).which is also referred to as Id3 (4). The HLH domain in

We assigned the ID1 gene to chromosome 20 by hy-all three genes is highly conserved.bridizing the plasmid containing the partial cDNA toSouthern blots of a PstI-digested somatic cell hybrid DNA1 To whom correspondence should be addressed.

385 GENOMICS 30, 385–387 (1995)0888-7543/95 $12.00

Copyright q 1995 by Academic Press, Inc.All rights of reproduction in any form reserved.

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panel. The probe showed signals syntenic with chromo-some 20, with 100% concordance (data not shown). FISHwas then used to refine the chromosomal localizationof the ID1 gene; a total of 39 metaphases with hybrid-ization signals were analyzed. The signals clustered onband 20q11. No specific double signals were observedon any other chromosomal regions, thus allowingthe precise localization of the ID1 gene to 20q11.(Fig. 1).

Southern blot analysis of the ID2 gene on the PstI-digested monochromosomal somatic cell hybridpanel gave signals with the NA10826B hybrid con-taining human chromosome 2 (data not shown). Atotal of 50 metaphases with FISH signals were ana-lyzed. Specific hybridization signals were clusteredat 2p25, thus allowing subregional assignment of FIG. 2. Localization of ID2 gene by fluorescence in situ hybridiza-

tion. (A) Partial metaphase showing specific site of hybridization ofID2 to 2p25 (Fig. 2).the ID2 gene. Arrow indicates FISH signal. (B) Partial metaphaseId proteins belong to an expanding family of eu-showing the DAPI-stained chromosome bands. Arrowhead indicateskaryotic transcriptional regulators characterized by band 2p25. (C) Idiogram of the human G-banded chromosome 2,

the presence of a highly conserved HLH motif, which illustrating the site of the ID2 gene hybridization. Each large starrepresents 10 double signals (on both chromatids), and each largemediates homo- and heterodimerization (9, 10). Idcircle represents 10 single (on one chromatid) signals. Small starsgenes are strongly expressed in undifferentiatedand circles represent one double and one single signal, respectively.proliferating and tumor cells. They can be induced

upon stimulation with serum, growth factors, orphorbol esters but are downregulated in quiescent

ACKNOWLEDGMENTScells or during differentiation (1–5, 8, 11). Id familyproteins are suggested to function as general inhibi-

This investigation was supported by NIH Grant CA-34775tors of differentiation (7) and controllers of growth(R.S.K.C.) and NSF Grant IBN-9118977 (R.B.).

induction. Also, the Id proteins are required for pro-gression through the G1 phase (6) and are involved

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