RESUMO
The MLL gene is frequently involved in chromosomal translocations associated with high-risk acute leukaemia. Infant and therapy-related acute leukaemia patients display chromosomal breakpoints preferentially clustered in the telomeric portion of the MLL breakpoint cluster region (SCII). Here, we demonstrate that SCII colocalizes with a gene-internal promoter element in the mouse and human MLL gene, respectively. The mRNA generated encodes an N-terminally truncated version of MLL that still exhibits many functional regions, including the C-terminal SET-domain. Etoposide-induced DNA double-strand breaks colocalize with the binding site of RNA polymerase II and the transcription initiation region, but not with a nearby Topo II consensus sequence. Thus, the observed genomic instability of the human MLL gene is presumably linked to transcriptional processes. The consequences of this novel finding for the creation of chromosomal translocations, the biology of the MLL protein and for MLL-mediated acute leukaemia are discussed.
Assuntos
Proteína de Leucina Linfoide-Mieloide/genética , Regiões Promotoras Genéticas , Recombinação Genética , Transcrição Gênica , Animais , Cromatina , Etoposídeo , Histona-Lisina N-Metiltransferase , Humanos , Camundongos , RNA Polimerase IIRESUMO
Chromosomal translocations t(4;11)(q21;q23) are associated with a group of acute lymphoblastic leukemias with very poor prognosis. From the complete sequences of the breakpoint cluster regions of the human MLL and AF-4 translocation partner genes, a novel set of 66 oligonucleotides that facilitates the rapid identification of translocation breakpoints by PCR analysis of genomic DNA was designed. For each breakpoint, a pair of optimally snited primers can be assigned, which improves the monitoring of the disease during treatment. Comparison of the breakpoints with the corresponding parental sequences also contributes to our better understanding of the illegitimate recombination events leading to these translocations.
Assuntos
Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 4/genética , DNA de Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição , Translocação Genética , Elementos Alu , Sítios de Ligação , Criança , Pré-Escolar , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 4/ultraestrutura , Análise Mutacional de DNA , Primers do DNA , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Proteína de Leucina Linfoide-Mieloide , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Fatores de Elongação da TranscriçãoRESUMO
Chromosomal translocations t(4;11) are regularly associated with a specific type of acute leukemias and probably initiate the development of this disease. It has been proposed by others, that these translocations are mediated by recombinases of the immune system. The breakpoints on both derivative chromosomes for three t(4;11) leukemia-derived cell lines and primary blasts from two patients have been analysed here in detail. The results revealed that: (a) multiple double- or single-stranded DNA breaks must have occured near the translocation breakpoints on both participating chromosomes; and (b) DNA fragments flanked by these breaks must have either been deleted, inverted or duplicated during the translocation process. We found no evidence for the involvement of specific target sequences and recombinases of the immune system. Similar characteristic features were observed by re-interpretation of published t(6;11) and t(9;22) translocation data. Therefore we present a new model for the generation of these translocations which poses, that these translocations are reciprocal but not balanced at the fine structure level and that the DNA damage-repair machinery is likely involved in producing the final structure of the translocation breakpoint.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 4 , Dano ao DNA , Reparo do DNA , DNA de Neoplasias , Proteínas de Ligação a DNA/genética , Leucemia/genética , Proteínas Nucleares/genética , Translocação Genética , Sequência de Bases , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 6 , Cromossomos Humanos Par 9 , Éxons , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Recombinação Genética , Fatores de Elongação da Transcrição , Células Tumorais CultivadasRESUMO
Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by 'error-prone-repair' mechanisms, but not by the action of recombinases of the immune system.
Assuntos
Dano ao DNA , Reparo do DNA , Leucemia de Células B/genética , Proto-Oncogenes , Fatores de Transcrição , Translocação Genética , Adolescente , Adulto , Sequência de Bases , Linfoma de Burkitt/etiologia , Linfoma de Burkitt/genética , Criança , Pré-Escolar , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 4 , Proteínas de Ligação a DNA/genética , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Leucemia de Células B/etiologia , Masculino , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/etiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Fatores de Elongação da TranscriçãoRESUMO
Derivative chromosomes of 40 patients diagnosed with t(4;11) acute lymphoblastic leukemia (ALL) were analysed on the genomic DNA level. Chromosomal breakpoints were identified in most cases within the known breakpoint cluster regions of the involved MLL and AF4 genes. Due to our current knowledge of the primary DNA sequences of both breakpoint cluster regions, specific features were identified at the chromosomal fusion sites, including deletions, inversions and duplications of parental DNA sequences. After separation of all t(4;11) leukemia patients into two age classes (below and above 1 year of age), the analysis of chromosomal fusion sites revealed significant differences in the distribution of chromosomal breakpoints and led to the definition of two hotspot areas within the MLL breakpoint cluster region. This may point to the possibility of different age-linked mechanisms that were leading to t(4;11) chromosomal translocations.
Assuntos
Quebra Cromossômica , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 4 , Proteínas de Ligação a DNA/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição , Adulto , Criança , Inversão Cromossômica , Reparo do DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Recém-Nascido , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Translocação GenéticaRESUMO
Chromosomal translocations t(4;11) are based on illegitimate recombinations between the human MLL and AF4 genes, and are associated with high-risk acute leukemias of infants and young children. Here, the question was asked, whether a correlation exists between the location of translocation breakpoints within both genes and the location of S/MARs. In "halo mapping experiments" (to define SARs), about 20 kb of MLL DNA was found to be attached to the nuclear matrix. Similar experiments performed for the translocation partner gene AF4 revealed that SARs are spanning nearly the complete breakpoint cluster region of the AF4 gene. By using short DNA fragments in "scaffold reassociation experiments" (to define MARs), similar results were obtained for both genes. However, Distamycin A competition experiments in combination with "scaffold reassociation experiments" revealed specific differences in the affinity of each tested DNA fragment to bind the isolated nuclear matrix proteins. When the latter data were compared with the known location of chromosomal breakpoints for both genes, an unexpected correlation was observed. DNA areas with strong MAR affinity contained fewer translocation breakpoints, while areas with weak or absent MAR affinity showed a higher density of chromosomal breakpoints.
Assuntos
Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 4/ultraestrutura , DNA de Neoplasias/metabolismo , Matriz Nuclear/metabolismo , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Quebra Cromossômica , Mapeamento Cromossômico , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 4/genética , Mapeamento de Sequências Contíguas , DNA Complementar/genética , DNA de Neoplasias/genética , Humanos , Proteína de Leucina Linfoide-Mieloide , Reação em Cadeia da Polimerase , Recombinação GenéticaRESUMO
Interleukin-2-deficient mice (IL-2(-/-)) manifest severe immune system abnormalities characterized by an uncontrolled activation and proliferation of lymphocytes. A systemic autoimmune syndrome results, and hemolytic anemia leads to early death especially in mice derived from a BALB/c genotype. Remarkably, IL-2 treatment prevents both the activation of the immune system and the development of autoimmune disease. Moreover, adoptive transfer of lymphocytes from IL-2-treated IL-2(-/-) animals confers protection to IL-2(-/-) mice, suggesting that IL-2 induces a postnatal differentiation/maturation of regulatory cells necessary for self- and non-self-discrimination.