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. 1998 Jul 21;95(15):8703-8.
doi: 10.1073/pnas.95.15.8703.

Positional cloning of ZNF217 and NABC1: genes amplified at 20q13.2 and overexpressed in breast carcinoma

C Collins  1 , J M Rommens, D Kowbel, T Godfrey, M Tanner, S I Hwang, D Polikoff, G Nonet, J Cochran, K Myambo, K E Jay, J Froula, T Cloutier, W L Kuo, P Yaswen, S Dairkee, J Giovanola, G B Hutchinson, J Isola, O P Kallioniemi, M Palazzolo, C Martin, C Ericsson, D Pinkel, D Albertson, W B Li, J W Gray
Affiliations

Positional cloning of ZNF217 and NABC1: genes amplified at 20q13.2 and overexpressed in breast carcinoma

C Collins et al. Proc Natl Acad Sci U S A. .

Abstract

We report here the molecular cloning of an approximately 1-Mb region of recurrent amplification at 20q13.2 in breast cancer and other tumors and the delineation of a 260-kb common region of amplification. Analysis of the 1-Mb region produced evidence for five genes, ZNF217, ZNF218, and NABC1, PIC1L (PIC1-like), CYP24, and a pseudogene CRP (Cyclophillin Related Pseudogene). ZNF217 and NABC1 emerged as strong candidate oncogenes and were characterized in detail. NABC1 is predicted to encode a 585-aa protein of unknown function and is overexpressed in most but not all breast cancer cell lines in which it was amplified. ZNF217 is centrally located in the 260-kb common region of amplification, transcribed in multiple normal tissues, and overexpressed in all cell lines and tumors in which it is amplified and in two in which it is not. ZNF217 is predicted to encode alternately spliced, Kruppel-like transcription factors of 1,062 and 1,108 aa, each having a DNA-binding domain (eight C2H2 zinc fingers) and a proline-rich transcription activation domain.

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Figures

Figure 1
Figure 1
(a) Physical map of 20q13.2 between D20S902 and WI-9227. Clones that make up the map are indicated as horizontal lines. The lengths of the lines are proportional to the sizes of the clones. The last five digits of the RMC identifiers listed in Table 1 identify clones. Mapped STSs and ESTs in this region are shown as vertical black and red lines, respectively. •, Clones that tested positive for STS or EST content by PCR. The approximate genome locations of five genes and a pseudogene are shown above the map. Arrowheads indicate transcriptional polarity where known. The approximate locations of 17 of 30 directly selected cDNA fragments are shown at the top. (b) FISH analysis of DNA sequence copy number in five informative breast tumors and one breast cancer cell line. Probes used in this study are indicated. The probe labeled COMP comprised clones P4030, P4040, P4038, P4039, P4041, and P4042. The vertical axis shows the number of hybridization signals produced by FISH with each probe. The number of signals is truncated at 20 because hybridization signal enumeration was difficult above this level. The x axis scales are the same for a and b. The results of six FISH studies are color coded by sample. Solid lines connect the measurements made for each sample. A ≈260-kb region of common maximal amplification is indicated by gray arrows above (a) and below (b).
Figure 2
Figure 2
Expression of ZNF217 and NABC1. (a) Hybridization of a ZNF217 probe to multiple tissue poly(A)+ Northern blots. (b) Northern blot analysis of ZNF217 transcription in three independent karyotypically normal breast epithelial cultures of finite lifespan (161RM, 48RM, 185RM), one immortalized line (184BS) and five breast cancer cell lines (MDA10, SKBR3, MDA436, BT474, MCF7). The breast cancer cell lines carry 4, 10–15, 10–15, >20, and >20 copies of the region encoding ZNF217, respectively. (c) Northern blot analysis of NABC1 transcription in the same cell lines. (d) Analysis of ZNF217 transcription in four paired breast tumor/normal epithelium samples. Lanes containing RNA from normal and tumor tissues are labeled N and T, respectively. (e) Analysis of NABC1 transcription by using the same membrane described in d. (f) QPCR analysis of ZNF217 mRNA abundance relative to that for GAPDH in 11 primary breast tumors and nine breast cancer cell lines. DNA sequence copy number at the ZNF217 locus determined by FISH with RMC20B4097 is indicated above each QPCR measurement.
Figure 3
Figure 3
Predicted amino acid sequences. (a) NABC1. (b) ZNF217. Eight putative C2H2 zinc finger domains are indicated in bold. A repeated motif in exon 1 is double underlined. Lines above the text indicate the positions of two putative tyrosine phosphorylation sites predicted by the PROSITE pattern search algorithm. A putative proline-rich transcription activation domain is encoded by amino acids 757-1005 and is composed of 16% proline. Exon boundaries were determined from the cDNA and genomic sequences and are marked with arrows. Exon 1 codes for 455 aa and exon 3 codes for 517 aa. The amino acid sequence of the alternately processed exon 4 is underlined.
Figure 4
Figure 4
Genomic organization of ZNF217. (A) The genomic organization of the five exons with encoded initiation and termination codons that make up ZNF217. Exon 4 encodes a TGA termination codon and is alternatively processed. Hatched boxes represent known 5′- and 3′-untranslated regions (UTR) in the cDNA. The sizes of exons and introns appear below and above the map, respectively. (B) The map of the 5632-bp ZNF217 cDNA. Vertical bars represent exon boundaries. The relative positions of the predicted eight C2H2 Kruppel-like zinc finger motifs are indicated by white circles. The position of the proline-rich putative transcription activator domain is shown as a hatched oval. AUUUA motifs are indicated in the 3′-untranslated region. The relative locations of three ESTs are shown in boxes.
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