LIM motif oncogenes

The LIM motif oncogenes are genes that are held responsible for the development of tumors in the immune system . The following article describes the investigation of a so-called breakpoint region in the case of T-cell leukemia and the discovery of the first candidate gene from the group of LIM motif oncogenes, the so-called rhombotin gene.

Since around the seventies, chromosomes have been shown in just as much detail in humans as in many other living beings; this was not possible before. This became aware of the fact that there were changes in the chromosomes in a large number of tumors. The chromosome alteration under discussion in this article is one in which pieces of chromosomes are swapped between two different chromosomes. This observation is very important because there are a number of tumors in which these chromosome changes are always in the same place.

Chronic myeloid leukemia is a very prominent example of such a situation . In CML, parts of chromosome 9 and chromosome 22 are exchanged for each other. This creates a typically altered chromosome called the Philadelphia chromosome . Over time, such changes were also seen in other forms of blood cancer . One of the most important findings was that similar chromosomal changes occurred in two different tumors. It was particularly noteworthy that these tumors occur in different species, but the principle of mutation is similar. These are Burkitt's lymphoma in humans and plasmacytoma in mice. In both cases the antibody genes were involved in the chromosome changes. The antibody genes are noteworthy because they also have small chromosome rearrangements in a narrow area, which are part of normal cell function and have nothing to do with tumor development.

On the basis of this finding, other tumors have been investigated in which, physiologically, there are also small, circumscribed chromosome rearrangements in certain genes. These are the cells of the immune system that mediate what is known as cellular immunity (this involves fighting viral infections but also rejecting transplants ). These cells (also called T lymphocytes ) rearrange a group of genes called T cell receptors . A T cell receptor is a protein that has a structure similar to an antibody . There are now tumors that arise from T cells. These are the so-called T-cell leukemias . In T-cell leukemia, too, it has now been found that there are rearrangements in the genes for the T-cell receptor, in which there was an exchange of large regions between different chromosomes, i.e. rearrangements of chromosome strands between different chromosomes.

In the 1980s, these areas of the chromosomes of the three diseases mentioned (CML, Burkitt's lymphoma and T-cell leukemia) were examined in such a way that the gene regions that corresponded to the breakpoints between the chromosome rearrangements were studied. Similar situations were found in all cases: genes were discovered that have been identified as oncogenes because they are held to be responsible for being significantly involved in tumor development. The present article describes in detail such a breakpoint analysis in T-cell leukemia.

The putative candidate gene on chromosome 11p13 and 11p15

In the laboratory of TH Rabbits in Cambridge , the mechanism of tumor development through chromosomal translocations in T-cell leukemia has been investigated using the example of the rhombotin gene. A number of consistent chromosomal translocations have been described in immature T cells. They affect the TCR-alpha-delta locus in 14q11 and the areas of 11p13 and 11p15. A T cell with the immature phenotype : CD3, CD4 and CD8 and the translocation t (11; 14) (p15; q11) has been characterized in a human T cell leukemia and the T cell line RPMI 8402 been. This cell rearranged and expressed TCR-beta and TCR-gamma, but shows no expression of the TCR-alpha-delta locus. The assumption was that, analogous to the findings in Burkitt's lymphoma and CML, a candidate gene is located in the area of chromosome 11p13-15 .

The translocating region at a glance

The investigated translocation t (11; 14) (p15; q1) lies immediately 3 'of a D-delta element of chromosome 14 and immediately 5' of a psi-rec signal of chromosome 11 in 14q and approx. 40 bp 5 'of a J-delta element of chromosome 14 and the region of chromosome 11 in 11p + which immediately adjoins the psi-rec signal. 3 'from the psi-rec of chromosome 11 lies a purine / pyrimidine alternating region, which represents a potential Z-DNA region, at the same distance as the J-delta elements on chromosome 14 from the corresponding breakpoint . Approx. 2kb 3 'from the breakpoint in chromosome 11 is a transcriptionally active region which codes for an approximately 4kb mRNA. In summary, one can say that the immature T-ALL described with the t (11; 14) translocation as an early thymocyte has attempted to rearrange the TCR delta locus, in which the TCR recombinase incorrectly gave a psi -rec signal on chromosome 11 in the immediate vicinity of a coding region.

Comparison of the breakpoints in different patients

In a further investigation step, four T-cell eukemias with the translocation t (11; 14) (p13; q11) were analyzed. The breakpoints of tumors 8508, 8511, 9989 and LAWL-2 clustered in a region of only 0.8 kb on chromosome 11p13, and were distributed on chromosome 14q11 in the entire approx. 100 kb area of the TCR-alpha / delta DJC tandem. The breakpoint of the translocation in tumor 8508 was in the TCR-delta locus near J-delta1, with no psi-rec signal at the breakpoint of 11p13, in tumor 8511 in the immediate vicinity of a pseudo-heptamer sequence of the Chromosome 11p13 and 5 'of J-delta2 of TCR-delta, and in tumor 9989 in the 3' half of the J-alpha region. In the case of the LALW-2 tumor, the breakpoint on chromosome 14 was about 1kb 5 'from J-delta1 in the immediate vicinity of D-delta2, which had apparently carried out a DD rearrangement with D-delta1, the breakpoint in 11p13 not being there psi-rec in the immediate vicinity. The translocation presumably resulted from an incorrect attempt by the early thymocyte to perform a VD rearrangement. An important conclusion from this study is the assumption that errors in the recombinase-mediated sequence-specific rearrangements in rearranging loci (TCR and Ig) cannot be the only cause of chromosomal translocations in lymphoid tumors.

Characterization of the breakpoint region: Z-DNA and DNase HSS

In a further comparative study of various breakpoint regions, the so-called "accessibility model" was modified. This model states that rearrangements only take place where the recombinase has access to the rec recognition sequences, and that this is made possible by a previous or simultaneous transcription of the corresponding rearrangement loci. So chromosomal translocations, which result from defective activity of the recombinase, could only take place in transcriptionally active areas. In the above-mentioned work, the translocations t (11; 14) (p13; q11), t (11; 14) (q13; q32) and t (7; 10) (q35; q24) were examined as possible exceptions. Potential Z-DNA regions were found in all three cases, in 10q24 of 32 bp in size, in 11p13 of 62 bp in length and in 11q13 several unusual purine / pyrimidine-alternating sequences (pu / py) of over 800 bp in length. In no case was transcriptional activity detected in the immediate vicinity, and in two cases, 10q24 and 11p13, DNase I hypersensitive regions could be detected. The breakpoint region was located between the potential Z-DNA region and the DNase I hypersensitive region. In addition, a coincidental connection between the bcr locus and the pu / py region could be largely excluded by demonstrating a high degree of interspecific sequence homology. These findings all suggest that the recombinase accessibility in the present cases is not mediated transcriptionally, but that changes in the chromatin structure allow the recombinase to access the rec signals.

The putative oncogene at the breakpoint t (11; 14) has an unusual transcription control

In a further investigation step, the structure of the coding sequence in 11p15 was investigated. It was found that the genomic locus of approx. 48 kb transcribed two different mRNAs from five exons (1.2 and 1.4 kb). However, the different messengers code for two almost identical proteins of 155 and 156 amino acids with a size of 17.7 kD. The amino acid sequence of exon 1 is: MVLDQED, of exon 1a: MMVLDKED. The interspecies homology mouse / human is approx. 98%. Preliminary expression studies showed that the 11p15 gene is preferentially expressed in neuroendocrine cells ( neuroblastoma , insulinoma , small-cell bronchial carcinoma - these are cells of the so-called APUD system) and that the various mRNA species are cell line-specific. No expression could be detected in lymphoid cells. In vitro CAT essays with sequences 5 'of exon 1 and 5' of exon 1a also showed that the 11p15 gene has two independent promoters for exon 1 and exon 1a, which, however, govern two practically identical proteins. Restriction recognition sites for the enzymes SacII BssHII, which are characteristic of so-called “methylation free islands”, were found in the promoter region . HpaII and MspI essays of thymus DNA showed that two approximately 0.5 kb regions 5 'of exons 1 and 1a are hypomethylated. Thus 11p15 belongs to a small group of genes (like the genes for alpha- amylase and the light chains of myosin ) in which an alternative splicing results in two practically identical proteins.

11p15 codes for a LIM domain protein

A sequence analysis of the 11p15 gene was u. a. based on the existence of 15 cysteines, suspect that this is a soluble, metal-binding protein that must have a fundamental function due to the great human / mouse homology . Further sequence analyzes showed that a region rich in cysteine / histidine in exon 2 of the 11p15 gene is completely conserved between humans and Drosophila . A good match with the cysteine-rich region of the genes lin-11 and mec-3 from Caenorhabditis elegans and the vertebral isl-1 gene brings together 11p15 to form the so-called LIM-domain genes (for lin1l isl-1 mec-3). However, 11p15 differs from the three above, which also have a 3 'close home domain. lin-11 is responsible for the asymmetrical division of a vulvar progenitor cell in worms, mec-3 is relevant in the cell differentiation of sensitive neurons of Caenorhabditis elegans and isl 1 codes for an insulin gene enhancer binding protein in rats. The lack of a DNA binding region suggests that 11p15 is a transcriptional regulator that functions through competitive protein dimerization .

11p15 is in the nervous system and the immune system expressed

The expression of the 11p15 gene in the embryonic development of the mouse was examined in two further studies . Four different experimental approaches were chosen for this. In Greenberg's work, a fusion construct from one of the two 11p15 promoters and the lacZ gene was used to produce transgenic mice. It was found that this construct is expressed in a segment-specific manner in the mouse brain stem . In a second approach, in situ hybridizations of complete mouse embryos, Northern blotting of RNA from embryonic tissue and a semiquantitative PCR analysis of the RNA obtained from embryonic tissue were carried out. The in situ hybridization of 11p15-antisense samples in the brain of newborn mice shows an expression of the rhombotin gene in the area of the caudate and putamen. Sagittal sections of 19-day-old mouse embryos show rhombotin expression in the entire area of the CNS, including the spinal cord, and low expression in the thymus. The following points are worth mentioning about other special features of the rhombotin family: The rhombotin (11p15) family contains at least three members and is involved in another T-cell translocation. Rhombotin2 is essential for the development of erythrocytes . Rhombotin and Myc are members of the network of transcription factors.

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