Characterization of a putative tumor suppressor region identified by the elimination test on human 3p21.3

Abstract

An experimental system, called the elimination test (Et), was developed to identify tumor growth antagonizing genes. Chromosome 3 containing microcell hybrids (MCHs) were passaged through SCID mice and regular deletion of the short arm of chromosome 3 (3p) were found by cytogenetic and molecular methods. The Et focused on 3p due to its frequent deletion in 23 tumor types including renal cell carcinoma (RCC), small cell lung carcinoma (SCLC) and breast cancer. By increasing the number of the tumors and by marker enrichment in the region of interest the common eliminated region I (CER1 or C3CER1) was reduced gradually to 1.6 cM on 3p21.3. Our aim was to further decrease the size of C3CER1 and identify the gene content. Additional markers were selected to further study the panel of MCH derived SCID tumors. We reduced C3CER1 to approximately 1 Mb and covered it with a PAC contig. Fiber-FISH was performed with PAC clones to assess the integrity of our contig.

From the telomeric part of C3CER1 two overlapping PACs were selected for sequencing. We identified a novel LIM domain containing gene, which we named to LIM domain containing 1 (LIMD1). We have characterized and mapped the mouse ortholog (Limd1) of the human gene. They both contain three tandemly arrayed LIM domains at their C-terminal ends. As the continuation of the gene identification in C3CER1, we constructed a physical and transcriptional map of a 250 kb region, which included four additional genes (LZTFL1, KIAA0851/SAC1, XT3, CCR9). We characterized a novel leucine zipper containing gene, the Leucine Zipper Transcription Factor- Like 1 (LZTFL1) and its mouse ortholog (Lztfl1). The LZTFL1 gene has two isoforms displaying alternative polyadenylation.

The further detailed description of the transcriptional content of the C3CER1 was possible due to further sequencing of eleven PAC clones and the appearance of new sequences in the public database. We assembled all the available data and detected 19 genes and 3 processed pseudogenes whithin a 1.4 Mb comprehensive transcriptional map. We identified and characterized four novel genes: FYVE and coiled-coil domain containing 1 (FYCO1), Transmembrane protein 7 (TMEM7), Leucine-rich repeat-containing 2 (LARRC2), Leucine Zipper Protein 3 gene (LUZP3). The refined centromeric border of C3CER1 is located inside the LRRC2 gene. A chemokine receptor cluster was recognized within C3CER1, which consisted of 8 genes.

The availability of the mouse sequence from Celera database made it possible to apply a comparative study to further investigate C3CER1 A 1.32-Mb human genomic sequence was compared with its 907-kb mouse orthologous sequence. The corresponding mouse region was found divided into two blocks, but their gene content and gene positions were highly conserved between human and mouse. We observed that two orthologous mouse genes (Xtrp3s1 and Cmkbr1) were duplicated. We also recognized a large number of conserved elements that were neither exons of known genes, CpG islands, nor repeats. We further identified and characterized five novel orthologous mouse genes (Kiaa0028, Xtrp3s1, Fyco1, Tmem7, and Lrrc2). The murine/human conservation breakpoint region (CBR) has features that characterize unstable chromosomal regions: deletions in YAC clones, late replication, gene and segment duplications, pseudogene insertions.

The detailed transcriptional map of C3CER1 was prerequisite for the delineation of its role in tumorigenesis. We found 18 genes within C3CER1, which will be studied further. Preliminary results indicate that the LTF and the LIMD1 are candidate rumor antagonizing genes.