As published in Cell Vol. 82, pp. 57-65, July 14, 1995

Disruption of the Architectural Factor HMGI-C:

DNA-Binding AT Hook Motifs Fused in Lipomas to Distinct Transcriptional Regulatory Domains

H.R. Ashar,*1 M. Schoenberg Fejzo,*2 A. Tkachenko,*1 X. Zhou,1 J.A. Fletcher,3,4,5,6 S. Weremowicz,3,6 C.C. Morton,3,6 and K. Chada1
1 Department of Biochemistry
Robert Wood Johnson Medical School
University of Medicine and Dentistry of New Jersey
Piscataway, New Jersey 08854
2 Department of Genetics
3 Department of Pathology
4 Department of Pediatrics
5 Division of Pediatric Oncology
Dana-Farber Cancer Institute
6 Department of Pathology
Brigham and Women's Hospital
Harvard Medical School
Boston, Massachusetts, 02115

* These authors contributed equally to this work.

Lipomas are one of the most common mesenchymal neoplasms in humans. They are characterized by consistent cytogenetic aberrations involving chromosome 12 in bands q14-15. Interestingly, this region is also the site of rearrangement for other mesenchymally derived tumors. This study demonstrates that HMGI-C, an architectural factor that functions in transcriptional regulation, has been disrupted by rearrangement at the 12q14-15 chromosomal breakpoint in lipomas. Chimeric transcripts were isolated from two lipomas in which HMGI-C DNA-binding domains (AT hook motifs) are fused to either a LIM or an acidic transactivation domain. These results, identifying a gene rearranged in a benign neoplastic process that does not proceed to a malignancy, suggest a role for HMGI-C in adipogenesis and mesenchyme differentiation.

Figure 1. Genomic Structure of the Human HMCI-C Gene

 

 

 

 

 

 

 

(A) H403, H409, H5003, H1001, and H4002 are genomic (lambda)FIXII clones (see Experimental Procedures) that contain the five exons (E1-E5) of the human HMGI-C gene. (B) Exons are denoted by boxes and introns by a line. Overlapping lambda clones were not obtained within intron 3, and this region is denoted with a broken line. Sequences encoding potential functional domains, AUG and UAG codons, are shown in the exons. The AT hook motifs of the DNA-binding domains are shown as stippled areas, and the closed region (in E5) encodes for the acidic domain of unknown function. The figure is not drawn to scale because of the large 5' and 3' untranslated regions.

 

Figure 2. FISH Mapping of HMGI-C lambda Clones to Metaphase Chromosomes from Three Lipomas Reveals Rearrangement of HMGI-C in All Three Tumors

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The normal chromosome 12 homologs provide internal positive hybridization controls and are marked by yellow arrows in each metaphase, while derivative chromosomes are marked by red arrows. Lambda clones H403 and H409 from the 5' end of HMGI-C were used as FISH probes to lipoma metaphase chromosomes from ST90-375 (A) and ST93-724 (C), respectively. Note hybridization on the normal chromosome 12 and the der(12), demonstrating that these clones map proximal to the breakpoint in both lipomas. In contrast, when H403 was hybridized to lipoma metaphase chromosomes from ST91-198 (E) hybridization was observed on the der(13) showing a map position distal to the breakpoint in this tumor. H4002 from the 3' end of HMGI-C was used as a FISH probe to lipoma metaphase chromosomes from ST90-375 (B) and ST93-724 (D); note hybridization on the normal chromosome 12 and the der(15) or der(3), respectivesly, indicating that these clones map distal to the breakpoint in both lipomas. However, FISH with H4002 from the 3' end of HMGI-C on ST91-198 (F) revealed hybridization on the normal chromosome 12 only, suggesting this clone is deleted from either the der(12) or der(13) in this tumor. Metaphase spreads were counterstained with DAPI. Lipoma karotypes are these: ST90-375, 46,XX,t(12; 15)(q15;q24); ST93-724, 46, XY, t(3; 12)(q29;q15); and ST91-198, 46,XX,t(12; 13)(q14-22;q21-32).

 

Figure 3. RT-PCR Amplification of HMGI-C Chimeric Transcripts

 

 

 

 

 

3' RACE on RNA from lipomas ST90-375 (375) and ST93-724 (724) yield 441 bp and 672 bp products, respectively. Reverse transcription was performed with an oligo(dT) primer linked to an adapter sequence and was followed by a nested PCR with sense primers from exon 1 and spanning exon 1 and 2. DLD-1 is a coloretal adenocarcinoma cell line that expresses wild-type HMGI-C (data not shown), but under these conditions, the predicted 3.1 kb wild-type message was not amplified. Products were analyzed on a 1.5% agarose gel. Lane M show molecular size markers in kilobases.

 

Figure 4. Rearrangements of 12q15 in Human Lipomas Disrupt the HMGI-C Gene and Produce Chimeric Transcripts

 

 

 

 

 

 

HMGI-C denotes the nucleotide and amino acid sequence of the wild-type gene, and the open box sequence corresponds to the end of HMGI-C exon 3. t(3;12) and t(12;15) refer to the nucleotide and predicted amino acid sequences of the chimeric transcripts from the cloned cDNA products obtained by a 3' RACE on RNA isolated from primary cell cultures of ST93-724 t(3;12) and ST90-375 t(12;15), respectively. Chr. 3 and chr. 15 refer to the novel sequences derived from chromosome 3 or 15 in t(3;12) and t(12;15) lipomas, respectively. Only the sequences immediately adjacent to the fusion sites are shown.

 

Figure 5. RT-PCR Using Primers Located on Either Side of the Fusion Site, between HMGI-C and Novel Sequences

 

 

 

 

 

RNA refers to the lipoma source of total RNA. Primer 375 is an oligonucleotide that is complimentary to the novel sequence from the chimeric transcript of lipoma ST90-375 and is located 8 nt downstream to the fusion point. Primer 724 is a complimentary oligonucletide to the novel sequence from the chimeric transcript of lipoma ST93-724 and is located 425 nt downstream of the fusion point. Total RNA from both lipoma primary cell cultures was reverse transcribed using either 375 or 724 primers and PCR amplified using HMGI-C sense primer (which spans exons 1 and 2) and the antisense primer used for reverse transcription. Expected product sizes are 180 bp from ST90-375 cDNA with 375 primer and 597 bp from ST93-724 cDNA with 724 primer.

 

Figure 6. Novel Sequences Fuesed to the DNA-Binding Domains of HMGI-C Encode Transcriptional Regulatory Domains

 

 

 

 

 

 

 

 

(A) Comparison of the novel chromosome 3 sequence from ST93-724 with the LIM domain-containing proteins zyxin (Sadler et al., 1992), apterous (Ap) (Cohen et al., 1992), Lh2 (Xu et al., 1993), LIN-11 (Freyd et al., 1990), and RBTN-1 (McGuire et al., 1989). Amino acids that constitute the LIM domain consensus are shown in bold. The amino acid spacing between residues is indicated by an x. In addition to the totally conserved cysteine, histidine, and aspartic acid residues (Sadler et al., 1992), LIM domains are characterized by the presence of an aromatic residue adjacent to the first histidine and a leucine located carboxy-terminal to the central HxxCxxCxxC cluster, which are indicated by arrows. Each LIM domain is designated 1, 2, or 3 depending on its position relative to the amino terminus.
(B) The potential transactivation acidic domain encoded by the sequence derived from chromosome 15 in ST90-375. Acidic residues are underlined and the amino acids serine and threonine are in bold.

 

Figure 7. Structure and Domain Organization of HMGI-C and the Predicted Fusion Proteins

 

 

 

 

The vertical broken line shows the location of junction sites in the chimeric products. DNA-binding domains of HMGI-C (AT) are preserved in the fusion proteins, but the carboxy-terminal domain (stippled) is replaced by potential transcriptional regulatory domains. LIM, LIM domain; minuses, acidic domain; S, T, serine/threonine-rich domain.